Compositions and methods for the delivery of nucleic acids

ABSTRACT

A nanosized complex includes siRNA and a compound comprising formula (I):

RELATED APPLICATION

This application claims priority from U.S. Provisional Application Nos.62/738,677, filed Sep. 28, 2018, and is a Continuation-in-Part of U.S.Ser. No. 14/743,298, filed Jun. 18, 2015, the subject matter of whichare incorporated herein by reference in their entirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant No. EB00489awarded by The National Institutes of Health and Grant NumberDGE-0951783, awarded by the National Science Foundation GraduateResearch Fellowship. The United States government has certain rights tothe invention.

BACKGROUND

RNA interference (RNAi) is a natural antisense mechanism that cellspossess to regulate the expression of genes at the mRNA level. Thisprocess relies on the ability of short fragments (19-23 nucleotides inlength) of double stranded small interfering RNA (siRNA) to recognizeand guide complementary mRNA transcripts into the RNA-induced silencingcomplex (RISC). Once translocated into the RISC complexes, the mRNAtranscripts are cleaved and ultimately degraded inside the cell,rendering them ineffective for translation into proteins. Meanwhile, thesiRNAs are preserved and constantly recycled for further silencingevents. The ability to synthetically design siRNAs against particularmRNA sequences has triggered numerous clinical and pre-clinical studiesto tailor RNAi induced silencing against a variety of disease-relatedgenetic transcripts.

Delivery of siRNA with nanoparticles is desirable in order to overcometheir susceptibility to serum nucleases and their ability to stimulatethe innate immune system upon intravenous injection. The use ofnanoparticles can also prevent the possibility of off-target sideeffects. Nanoparticles are also required for successful siRNA therapiesbecause they can facilitate intracellular release following uptake bytumor cells. Endocytosis has been identified as a dominant cellularuptake mechanism, whereby foreign material delivered to cells will besequestered into endosomal compartments and ultimately degraded afterfusion with lysosomes. As a result, siRNA therapy faces an additionaldelivery barrier because access to the RISC complexes in the cytoplasmis necessary for effective treatment. Viral vectors have evolved formillions of years to become efficient carriers at introducing theirgenetic material into host cells. As a result, adenoviruses andretroviruses are currently explored as possible gene therapy vectors,but the success they have shown in vivo is masked by their potential toseverely trigger the immune response. Compared to viral vectors,non-viral or synthetic vectors have many advantages, such as lowimmunogenicity, low production cost, and ease of modification, thusmaking them very attractive for siRNA delivery platforms. However, muchresearch is still required in order to improve upon the low transfectionefficiencies of most current non-viral vectors.

Cationic lipid constructs are widely used as alternatives to viralnanoparticles. They form nanoparticles through the electrostaticinteraction with the negatively-charged siRNA. Such nanoparticles, orlipoplexes, can be designed to exhibit similar characteristics andbehaviors to those observed with viral vectors by utilizing pH sensitivemoieties that facilitate endosomal escape. This is typicallyaccomplished by incorporating an amine-rich head group with an overallslightly acidic pKa into the cationic lipid structure. Once they becomeprotonated in the acidic endosomal-lysosomal compartments, cationiclipoplexes are able to participate in membrane fusion and degradationevents by inducing an electrostatic flip-flop reorganization of anionicphospholipids in the membrane bilayer. This extensive transfer of lipidsneutralizes the charge interactions that govern particle formation,causing the inevitable dissociation of siRNA from the lipoplexes,followed by and subsequent release into the cytoplasm.

SUMMARY

Embodiments described herein relate to compounds used to formmultifunctional pH-sensitive carriers that are designed to condensenucleic acids and deliver the condensed nucleic acids to cells. Thecompounds can include a protonable amino head group, which can complexwith nucleic acids, fatty acid or lipid tails, which can participate inhydrophobic condensation, and two cystenyl residues capable of formingdisulfide bridges via autooxidation.

In some embodiments, the compound includes formula (I):

-   -   wherein R¹ is an akylamino group or a group containing at least        one aromatic group; R² and R³ are independently an aliphatic        group or a hydrophobic group derived, for example, from a fatty        acid; R⁴ and R⁵ are independently H, a substituted or        unsubstituted akyl group, an akenyl group, an acyl group, an        aromatic group, polymer, a targeting group, or a detectable        moiety; a, b, c, and d are independently an integer from 1 to 10        (e.g., a, b, c, and d are each 2); and pharmaceutically        acceptable salts thereof.

In some embodiments, R¹ can include at least one of:

-   -   where R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are        independently hydrogen, an alkyl group, a hydrophobic group, or        a nitrogen containing substituent; and e, f, g, i, j, k, and 1,        are an integer from 1 to 10. For example, R¹ can include at        least one of CH₂CH₂NH₂, CH₂CH₂NHCH₂CH₂NHCH₂CH₂NH, or        CH₂CH₂NHCH₂CH₂CH₂CH₂NHCH₂CH₂CH₂NH.

In other embodiments, R² and R³ are independently a hydrophobic groupderived from oleic acid or linoleic acid and are the same or different.

In some embodiments, R⁴ and R⁵ are independently H, a substituted orunsubstituted polymer, a targeting group, or a detectable moiety.

In some embodiments, the compound can have the formulas:

-   -   or pharmaceutically acceptable salts thereof.

In other embodiments, a targeting group can be covalently attached tothe compound by a linker. The targeting group can be a peptide, aprotein, an antibody, or an antibody fragment. The linker can include apolyamino acid group, a polyalkylene group, or a polyethyelene glycolgroup. The linker can also include an acid labide bond that ishydrolyzable in an endolyssomal environment following uptake to cells,such as cancer cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a synthetic procedure for ECO.

FIGS. 2(A-D) illustrate a schematic, plots, and immunoblots showingECO/siβ3 nanoparticles induced sustained gene silencing of β3 integrin.A) ECO forms nanoparticles with siRNA through electrostaticinteractions, disulfide cross-linking and hydrophobic interactions. B)β3 integrin mRNA expression in quiescent or TGF-β stimulated (5 ng/mL,72 hours) NME and MDA-MB-231 cells with the indicated treatment groupsat 100 nM siRNA by semi-quantitative real-time PCR (n=3, mean±SE, p≤0.01for all time points beyond 8 hours). Western blot analysis of β3integrin expression in quiescent or TGF-β stimulated (5 ng/mL, 72 hours)NME c) and MDA-MB-231 d) cells at the indicated time pointspost-nanoparticle treatment with the indicated treatment groups.

FIGS. 3(A-E) illustrate images, graphs, and an immunoblot showingECO/siβ3 nanoparticles attenuated TGF-β-mediated EMT, invasion andproliferation. A) Immunofluorescent images of actin cytoskeletonvisualized with rhodamine-conjugated phalloidin in mouse NME cells withdifferent treatments (scale bar, 100 m; inset scale bar, 50 m). B)Semi-quantitative real-time PCR analysis (n=3) of EMT markers in NMEcells (**p≤0.01). C) Western blot analysis of E-cadherin and N-cadherinin NME cells. D) Invasion assay of quiescent or TGF-β stimulated NMEcells (n=3, *p≤0.05, **p≤0.01). E) Proliferation as measured by[³H]thymidine incorporation of either quiescent or TGF-β stimulated NMEcells (n=3, *p≤0.05, **p≤0.01). For all experimental groups, NME cellswere pre-treated with TGF-β (5 ng/mL; 72 hours) followed by ECO/siRNAnanoparticle treatment using 100 nM siRNA. For panels b-e, datarepresent mean±SE. Results for panels c-e are representative of threeindependent experiments.

FIGS. 4(A-C) illustrate images and graphs showing ECO/siβ3 nanoparticlesattenuated 3D organoid outgrowth. NME and MDA-MB-231 cells were grown ina compliant 3D-organotypic microenvironment and treated with ECOnanoparticles containing Alexa Fluor 488-labeled siRNA. Cellular uptakeof ECO/siRNA nanoparticles monitored by fluorescence confocal microscopy(scale bar, 100 m). A) Bright-field microscopic image of a singleorganoid and fluorescence confocal microscopic images of ECO/siRNAnanoparticle uptake in the organoid over the course of 24 hours. B) NMEand c) MDA-MB-231 cells were grown in a compliant 3D-organotypicmicroenvironment for up to 10 days with or without prior TGF-βstimulation (5 ng/mL) for 72 h. On day 4, 6 and 8, cells were treatedwith ECO/siNS or ECO/siβ3 nanoparticles at 100 nM siRNA. Organoid growthat day 10 was monitored via longitudinal bioluminescence (n=4, *p≤0.05,**p≤0.01). For panels c-d, data represent mean±SE.

FIGS. 5(A-E) illustrate images and a graph showing RGD modification ofECO/siRNA nanoparticles enhances uptake in post-EMT breast cancer cells.A) confocal microscopy (scale bar, 50 μm) and B) quantified by flowcytometry (n=3, **p≤0.01). Quantitative analysis of β3 integrin mRNAlevels following treatment with siβ3 nanoparticles by real-time PCR(n=3, **p≤0.01) in (C) NME and (D) MDA-MB-231 cells revealedRGD-targeted ECO/siRNA nanoparticles maintain gene silencing. E)Cellular uptake in NME cells, both with and without TGF-β stimulation (5ng/mL; 72 hours), was quantified by flow cytometry for RGD-ECO/siRNAnanoparticles containing Alexa Fluor 488-labelled siRNA 4 hours aftertreatment. One group of TGF-β stimulated NME cells (TGF-β+ECO/siβ3) wastreated with ECO/siβ3 nanoparticles at 100 nM siRNA for 48 hours priorto cellular uptake with the RGD-targeted nanoparticles to quantify theeffect of β3 integrin silencing on targeted uptake (n=3, ±SE, *p≤0.05,**p≤0.01). For panels B-E, data represent mean±SE.

FIGS. 6(A-F) illustrate RGD-targeted ECO/siβ3 nanoparticles inhibitedprimary tumor growth and EMT in mice after systemic administration. A)and quantified by B) BLI (data represents mean±SE, n=5, *p≤0.05,**p≤0.01), and C) caliper measurements (data represents mean±SE, n=5,*p≤0.05, **p≤0.01). D) Primary tumors were resected at week 9, and finaltumor weights of the indicated treatment groups were obtained (datarepresents mean±SE, n=5, **p≤0.01). E) Semi-quantitative real-timequantification of β3 integrin mRNA expression from resected primarytumors of the indicated groups (data represents mean±SE, n=5, **p≤0.01).F) H&E, DAPI and fibronectin immunostaining of the indicated primarytumors (scale bar, 300 m).

FIGS. 7(A-E) illustrate RGD-ECO/siβ3 nanoparticles inhibited breastcancer metastasis and primary tumor recurrence. A) BLI images of mice atweek 12 revealed differences in metastasis and primary tumor recurrencefor the different treatment groups after primary tumor resection on week9. B) Quantification of primary tumor recurrence (data representsmean±SE, n=5, *p≤0.05). C) Quantification of thoracic metastasis by BLI(data represents mean±SE, n=5, *p≤0.05, **p≤0.01). Mice were releasedfrom ECO/siRNA therapeutic regimen at week 12. D) Representative BLI ofmice on week 16. E) Quantification of whole body tumors from D. (datarepresents mean±SE, n=5, *p≤0.05).

FIGS. 8(A-C) illustrate plots showing zeta potential of A) ECO/siRNA, B)PEG-ECO/siRNA, and C) PEG(HZ)-ECO/siRNA nanoparticles incubated in PBSsolutions at pH levels corresponding to stages of intracellulartrafficking (pHs 7.4, 6.5, 5.4).

FIG. 9 illustrates a plot showing the comparison of hemolytic activityof ECO, PEG-ECO, and PEG(HZ)-ECO siRNA nanoparticles at pH levelscorresponding to stages of intracellular trafficking. Relative hemolyticactivity was calculated with respect to the hemolytic activity of 1%Triton-X-100.

FIGS. 10(A-C) illustrate a graph, plot, and image showing A) Cellularuptake of unmodified, PEG-, PEG(HZ)-, RGD-PEG-, and RGD-PEG(HZ)-modifiedECO/siRNA nanoparticles quantified with flow cytometry using anAF647-labeled siRNA. B) Luciferase silencing of unmodified, PEG-,PEG(HZ)-, RGD-PEG-, and RGD-PEG(HZ)-modified ECO/siRNA nanoparticles inMDA-MB-231-luc triple-negative breast cancer cells. C) Confocalmicroscopy images of MDA-MB-231 cells incubated with RGD-PEG-, andRGD-PEG(HZ)-modified ECO/siRNA nanoparticles at 10 min, 3 hr, and 6 hr.DAPI, cell nucleus (blue); Lysotracker DND-26, lysosomes (green); siRNA,AF-647 (red).

FIGS. 11(A-D) illustrate in vivo luciferase silencing efficiencyfollowing a single i.v. treatment with various surface-modifiedECO/siRNA nanoparticles (1.0 mg/kg siRNA dose). A) Quantification ofbioluminescence signal from ROIs drawn over the tumor. B) RepresentativeBLI images of the different treatment groups. Tumor accumulation andretention of surface-modified ECO/siRNA nanoparticles following i.v.administration. C) Representative FMT images of a single mouse from eachtreatment group over 24 hours post-treatment with nanoparticlesformulated with an AF647-tagged siRNA. An ROI was drawn over the areacontaining the tumor. D) Quantification of fluorescence signal from eachROI. Flow cytometry and confocal microscopy analysis of single cellsuspensions obtained from primary MDA-MB-231 mammary fat pad tumorsfollowing i.v. administration of various surface modified ECO/siRNAnanoparticles.

FIGS. 12(A-C) illustrate evaluation of eIF4E mRNA and protein expressionas determined by A) qRT-PCR and B) western blot analysis in MDA-MB-231and MDA-MB-231.DR cells 5 days following treatment withRGD-PEG(HZ)-ECO/siRNA nanoparticles (N/P=8) delivering either sieIF4E orsiNS (100 nM). C) Dose-response curves as determined by MTT assay ofMDA-MB-231 and MDA-MB-231.DR cells treated with varying concentrationsof PTX following prior treatment with RGD-PEG(HZ)-ECO/siRNAnanoparticles delivering sieIF4E or siNS. Cells were first treated withRGD-PEG(HZ)-ECO/siRNA nanoparticles for 48 hours followed by treatmentwith varying concentrations of PTX for an additional 48 hours.

FIGS. 13(A-F) illustrate in vivo efficacy of combination therapyinvolving PTX and RGD-PEG(HZ)-ECO/sieIF4E nanoparticles. Alternatingtreatment of siRNA nanoparticles and PTX every 6 days began after 4weeks once the primary tumors reach an average of 150 mm³. A)Quantification of bioluminescent imaging over the course of theexperiment (data represents mean±SE, n=5, *p≤0.05, **p≤0.01) and B) BLIimages at week 10. C) Tumor growth was monitored using digital calipermeasurements (data represents mean±SE, n=5, *p≤0.05, **p≤0.01). D)Primary tumors were resected at week 10 and E) final tumor weights wereobtained (data represents mean±SE, n=5, *p≤0.05, **p≤0.01). F)Semi-quantitative real-time quantification of eIF4E mRNA expression fromthe resected primary tumors (data represents mean±SE, n=5, *p≤0.05,**p≤0.0, # p>0.05).

FIGS. 14(A-B) illustrate a schematic representation of formation andworking of RGD-PEG-ECO/siRNA nanoparticles. (a) Amino lipid carrier ECO(E) is mixed with the targeting moiety RGD-PEG-Mal followed byelectrostatic condensation with siRNA molecules. Oxidation of cysteineresidues (C) to form disulfide bonds and hydrophobic condensation of theoleic acid tails (O) further stabilize the formation ofRGD-PEG-ECO/siRNA nanoparticles. (b) RGD-PEG-ECO/siRNA nanoparticlesmediate efficient siRNA delivery by the PERC mechanism. Nanoparticlesare internalized into cells by receptor-mediated endocytosis and travelthrough the intracellular trafficking pathway. As the pH in theendosomes decreases, the pH-sensitive amphiphilicity of thenanoparticles enables them to induce endosomal membrane destabilizationand endosomal escape into the cytosol. The nanoparticles then undergoreductive dissociation, releasing the siRNA cargo into the cytoplasmwhere it encounters the RNAi machinery to silence the target IncRNA.

FIGS. 15(A-H) illustrate plots and an immunoblot showing DANCR issignificantly overexpressed in TNBC. (a) Endogenous expression of DANCRis significantly elevated in all breast cancer cell lines: MCF7 (luminalA; ER⁺, PR^(+/−), Her2⁻), ZR-75 (luminal B, ER⁺, PR^(+/−), Her2⁺), andHs578T, BT549, and MDA-MB-231 (claudin-low ER⁻, PR⁻, Her2⁻), compared toHMECs, and even more so in the TNBC cell lines. (b) Quantification ofDANCR expression from a breast cancer array containing cDNA from normalsubjects (n=4) and breast cancer patients shows that DANCR is highlyupregulated in tumor samples from TNBC patients (n=12), in comparisonwith breast tissues from normal subjects. (c) Differential geneexpression analysis for DANCR performed for 104 normal and 790 breastcancer samples from TCGA database shows a significant upregulation ofDANCR in breast cancer samples, particularly in 80 TNBC samples (d),compared to normal levels. (e) Heat map comparing and demonstrating theheterogeneity of DANCR expression in the individual normal (N) and TNBC(T) samples from TCGA database. DANCR expression is significantly higherin TNBC samples than breast cancer samples expressing (f) ER (n=584),(g) PR (n=508), and (h) HER2 (n=128) receptors (error bars denotes.e.m., *p≤0.05, ***p≤0.0005).

FIGS. 16(A-F) illustrate plots and images showing RGD-PEG-ECO/siDANCRnanoparticles mediate robust and sustained silencing of DANCR expressionin MDA-MB-231 and BT549 cells. RGD-PEG-ECO/NC and RGD-PEG-ECO/siDANCRnanoparticles were formulated at N/P=10, RGD-PEG/ECO=2.5 mol %, and[siRNA]=100 nM and characterized on LiteSizer. The particles show (a)and (b) single intensity peaks indicating uniform distribution, with (c)predicted size of about 108 nm and zeta potential of 22 mV. (d) Gelretardation assay of RGD-PEG-ECO/NC and RGD-PEG-ECO/siDANCRnanoparticles shows high encapsulation efficiency, with free siRNA ascontrol. (e) Morphology of RGD-PEG-ECO/NC and RGD-PEG-ECO/siDANCRnanoparticles is visualized by transmission electron microscopy. (f)MDA-MB-231 and BT549 cells transfected with RGD-PEG-ECO/NC andRGD-PEG-ECO/siDANCR nanoparticles were harvested on days 1, 4, and 7,for measurement of DANCR knockdown by qRT-PCR. DANCR expression bysiDANCR treatment is normalized to NC treatment for both the cell lines(scale bar=200 nm, error bars denote s.e.m., **p≤0.00005, *p≤0.005).

FIGS. 17(A-H) illustrate graphs and images showing RGD-PEG-ECO/siDANCRnanoparticles inhibit TNBC invasion, migration, and proliferation. (a)siDANCR-treated MDA-MB-231 and BT549 cells show 80-90% knockdown inDANCR expression after 24 h, compared to NC-treated cells (**p<0.0005).(b) DANCR silencing leads to substantial reduction in the invasivepotential of MDA-MB-231 and BT549 cells plated in Matrigel-coatedTranswell inserts in serum-free media for 24 h. The migrated cells werefixed with 10% formalin, stained with 0.05% crystal violet, and countedusing ImageJ software (**p<0.0005). (c & d) Standard scratch-woundassays demonstrate that siDANCR-treated MDA-MB-231 and BT549 cells havereduced ability to close the scratched wounds in 24 h, while theNC-treated cells readily migrate and close the wounds. (e) MTT assaydemonstrates significant reduction in cell viability of MDA-MB-231 andBT549 cells treated with siDANCR nanoparticles, compared to NC(*p<0.01). (f) DANCR silencing in MDA-MB-231 and BT549 cells results insignificant decrease in cell proliferation, as indicated by thedecreased BrdU incorporation, in comparison to NC (**p<0.0001). (g)MDA-MB-231 and BT549 cells treated with RGD-PEG-ECO/siDANCRnanoparticles show considerable reduction in their ability to form 3Dtumor spheroids, compared to NC-treated cells. (h) Clonogenic assayshows significant suppression of proliferative potential of MDA-MB-231and BT549 cells treated with RGD-PEG-ECO/siDANCR nanoparticles, comparedto NC nanoparticles (*p<0.01). Colonies formed after plating 2000siDANCR- and NC-treated cells were stained with 0.01% crystal violetafter 10 days, counted, and represented as % of survived colonies. Errorbars denote s.e.m., scale bar=100 μm.

FIGS. 18(A-E) illustrate immunoassays, graphs, and images showingRGD-PEG-ECO/siDANCR nanoparticles suppress the expression of proteinsinvolved in multiple oncogenic pathways. (a) DANCR silencing inMDA-MB-231 and BT549 cells induces significant decrease in theexpression of β-catenin (Wnt signaling); ZEB 1 and N-cadherin (EMTmarkers); and the anti-apoptotic protein, survivin. β-Actin is used asthe loading control. (b) RGD-PEG-ECO/siDANCR nanoparticle-mediatedsilencing of DANCR in MDA-MB-231 and BT549 cells downregulates theexpression of the PRC2 complex proteins EZH2 and SUZ12, along withdecreased H3K27 tri-methylation marks. (c) DANCR regulates PRC2 complexby directly binding to EZH2 in MDA-MD-231 and BT549 cells (*p<0.005).(d) Phospho-kinase arrays demonstrate the differential phosphorylationpatterns of 43 kinases with treatment of RGD-PEG-ECO/siDANCRnanoparticles in MDA-MB-231 and BT549 cells. Proteins highlighted in theboxes show significant differences in phosphorylation status between NCand DANCR knockdown. (e) The bar graphs represent the correspondingkinases/proteins that show significant changes (p<0.05) in expressionbetween control and DANCR silencing, calculated by comparing the meanpixel intensities of the duplicate spots.

FIGS. 19(A-I) illustrate plots, images, and graphs showing systemictherapy of RGD-PEG-ECO/siDANCR nanoparticles significantly reducesprimary tumor burden in TNBC xenografts. RGD-PEG-ECO/NC andRGD-PEG-ECO/siDANCR nanoparticles were formulated at N/P=8,PEG-RGD/ECO=2.5 mol % and [siRNA]=0.3 mg/mL, and characterized for (a)particle size and (b) zeta potential (*p<0.05). (c) Weekly intravenousinjections of RGD-PEG-ECO/siDANCR nanoparticles at a siRNA dose of 1.0mg/kg for 6 weeks result in significant reduction in tumor volumes(*p<0.05). MDA-MB-231 and BT549 xenografts (2 & 4×10⁶ cells,respectively) were implanted into the mammary fat pads of athymic miceand treatment was started at average tumor volume of 70-90 mm³. (d)Compared to RGD-PEG-ECO/NC, RGD-PEG-ECO/siDANCR nanoparticles mediatesignificant inhibition of tumor progression. (e) Tumor status at the endof 6 weeks. (f) H&E staining of paraffin-fixed tumor samples from micetreated with RGD-PEG-ECO/siDANCR and NC (scale bar=100 μm) shows largezonal necrosis (arrows) in NC but only focal necrosis with siDANCRtreatment. Compared to RGD-PEG-ECO/NC-treated tumors,RGD-PEG-ECO/siDANCR-treated tumors demonstrate (g) significantly reducedDANCR levels (**p<0.005), and significantly lower mRNA expression of (h)EZH2 and (i) survivin, indicating that the targeted RGD-PEG-ECO-siDANCRtherapy likely causes epigenetic alterations and apoptosis in the TNBCtumors (*p<0.05).

FIGS. 20(A-D) illustrate a plot and images showing systemicadministrations of RGD-PEG-ECO/siDANCR nanoparticles do not causeevident side-effects in the treated mice. (a) Both siDANCR- andNC-treated mice show no changes in their general health and nosignificant differences in the body weights during the 6 weeks ofnanoparticle injections. H&E staining of the tumor sections demonstratesno significant changes in the morphology of the vital organs in NC- andsiDANCR-treated mice, with the (b) liver, (c) kidney, and (d) lungsshowing normal hepatocyte, renal, and alveolar histology, respectively.(error bars denote s.e.m., scale bar=100 μm).

FIG. 21 illustrate images showing RGD-PEG-ECO/siDANCR nanoparticlesinhibit TNBC proliferation and survival. Clonogenic assay showssignificant suppression of proliferative potential of MDA-MB-231 andBT549 cells treated with RGD-PEG-ECO/siDANCR nanoparticles, compared toNC nanoparticles. Colonies formed after plating of 2000 siDANCR- andNC-treated cells were stained with 0.01% crystal violet after 7-10 days.

FIG. 22 illustrate a graph showing systemic administration ofRGD-PEG-ECO/siDANCR nanoparticles significantly reduces primary tumorburden in TNBC xenografts. Combination plot showing the final tumorvolumes (bars) and the individual tumor volumes (dots) of NC- andsiDANCR-treated mice bearing MDA-MB-231 and BT549 xenografts at 6 weekspost-treatment (error bars denote s.e.m., *p<0.05).

FIG. 23 illustrates a graphs showing BANCR expression in MCF-7 and DU145cell lines and their corresponding drug-resistant cell lines. (datarepresent mean±S.D., **p<0.01, ***p<0.001).

FIGS. 24(A-C) illustrate plots and a graph showing formulation ofECO/siRNA nanoparticles. (a) Size distribution of ECO/siRNAnanoparticles determined by DLS. (b) Zeta potential distribution ofECO/siRNA nanoparticles determined by DLS. (c) BANCR expression MCF-7-DRand DU145-DR cells after treatment with ECO/siNS and ECO/siRNAnanoparticles (data represent mean±S.D., n=3, **p<0.01).

FIGS. 25(A-C) illustrate a graphs and a plot showing Expression ofHOTTIP (a) and HOXA13 (b), and correlation between HOTTIP and HOXA13 (c)in different PCa cell lines (data represent mean±S.D., *p<0.05,**p<0.01, ***p<0.001).

FIG. 26 illustrates a plot showing correlation between HOTTIP and HOXA13in PCa tissues.

FIGS. 27(A-B) illustrate an image showing morphology of PC3 (a) andDU145-DR (b) cells.

FIGS. 28(A-D) illustrate plots and an immunoassay showing formulation ofECO/siRNA nanoparticles. (a) Size distribution of ECO/siRNAnanoparticles determined by DLS. (b) Zeta potential distribution ofECO/siRNA nanoparticles determined by DLS. (c) Gel retardation assay ofECO/siRNA nanoparticles. (d) HOTTIP expression PCa cells after treatmentwith ECO/siNS and ECO/siRNA nanoparticles (data represent mean±S.D.,n=4, *p<0.05).

FIGS. 29(A-B) illustrate graphs showing cell viability of PC3 (a) andDU145-DR (b) cells treated with or without ECO/siRNA nanoparticles atday 1 to 3 (data represent mean±S.D., *p<0.05, ***p<0.001).

FIG. 30 illustrates images showing Live/dead staining of PC3 andDU145-DR cells treated with or without ECO/siRNA nanoparticles at day 3.

FIG. 31 illustrates a graph showing cell viability of PC3 cells treatedwith different dose of ECO/siRNA nanoparticles at day 1 (data representmean±S.D., *p<0.05, ***p<0.001).

FIG. 32 illustrates images showing 3D tumor spheroid growth in Matrigelof PC3 and DU145-DR cells treated with or without ECO/siRNAnanoparticles at day 7.

FIG. 33 illustrates images showing cell migration ability of PCa cells(PC3 and DU145-DR) after treatment with ECO/siNS and ECO/siHOTTIPnanoparticles assessed by standard scratch-wound assays.

FIG. 34 illustrates images showing cell migration and invasion abilityof PCa cells (PC3 and DU145-DR) after treatment with ECO/siNS andECO/siHOTTIP nanoparticles assessed by Transwell study.

FIG. 35 illustrates Western blot images of EMT markers in PCa cells (PC3and DU145-DR) after treatment with ECO/siNS and ECO/sieIF4Enanoparticles.

FIG. 36 illustrates a graph showing expression of HOTTIP and EMT makersin DU145 cells treated with or without TGF-β1 at mRNA level (datarepreset mean±S.D., n=4, *p<0.05, **p<0.01).

FIG. 37 illustrates images showing cell migration and invasion abilityof DU145-TGF-β1 cells after treatment with ECO/siNS and ECO/siHOTTIPnanoparticles assessed by Transwell study.

DETAILED DESCRIPTION

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises, such as Current Protocolsin Molecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates). Unlessotherwise defined, all technical terms used herein have the same meaningas commonly understood by one of ordinary skill in the art to which thepresent invention pertains. Commonly understood definitions of molecularbiology terms can be found in, for example, Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th Ed., Springer-Verlag: New York,1991, and Lewin, Genes V, Oxford University Press: New York, 1994. Thedefinitions provided herein are to facilitate understanding of certainterms used frequently herein and are not meant to limit the scope of thepresent invention.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a pharmaceutical carrier” includes mixtures of two or moresuch carriers, and the like. “Optional” or “optionally” means that thesubsequently described event or circumstance can or cannot occur, andthat the description includes instances where the event or circumstanceoccurs and instances where it does not. For example, the phrase“optionally substituted lower alkyl” means that the lower alkyl groupcan or cannot be substituted and that the description includes bothunsubstituted lower alkyl and lower alkyl where there is substitution.

The term “alkenyl group” is defined herein as a C2-C20 alkyl grouppossessing at least one C═C double bond.

The term “alkyl group” as used herein is a branched or unbranchedsaturated hydrocarbon group of 1 to 25 carbon atoms, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl,heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and thelike. A “lower alkyl” group is an alkyl group containing from one to sixcarbon atoms.

The term “acyl” group as used herein is represented by the formulaC(O)R, where R is an organic group such as, for example, an alkyl oraromatic group as defined herein.

The term “alkylene group” as used herein is a group having two or moreCH₂ groups linked to one another. The alkylene group can be representedby the formula (CH₂)_(a), where a is an integer of from 2 to 25.

The term “aromatic group” as used herein is any group containing anaromatic group including, but not limited to, benzene, naphthalene, etc.The term “aromatic” also includes “heteroaryl group,” which is definedas an aromatic group that has at least one heteroatom incorporatedwithin the ring of the aromatic group. Examples of heteroatoms include,but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. Thearyl group can be substituted or unsubstituted. The aryl group can besubstituted with one or more groups including, but not limited to,alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone,aldehyde, hydroxy, carboxylic acid, or alkoxy.

The terms “cancer” or “tumor” refer to any neoplastic growth in asubject, including an initial tumor and any metastases. The terms“cancer cell” or “tumor cell” can refer to cells that divide at anabnormal (i.e., increased) rate.

The phrase “nitrogen containing substituent” is defined herein as anyamino group. The term “amino group” is defined herein as a primary,secondary, or tertiary amino group. In the alternative, the nitrogencontaining substituent can be a quaternary ammonium group. The nitrogencontaining substituent can be an aromatic or cycloaliphatic group, wherethe nitrogen atom is either part of the ring or directly or indirectlyattached by one or more atoms (i.e., pendant) to the ring. The nitrogencontaining substituent can be an alkylamino group having the formulaRNH₂, where R is a branched or straight alkyl group, and the amino groupcan be substituted or unsubstituted.

The term “long non-coding ribonucleic acid”, “long non-coding RNA” or“lncRNA” refers to a ribonucleic acid sequence that is encoded within agenomic intronic or intergenic region. Such lncRNAs are not transcribedinto proteins but act directly to regulate various activities including,but not limited to, transcription or translation. For example, an IncRNAmay be exemplified by an aptamer that regulates transcription rates of aparticular gene or allele.

The term “nucleic acid” refers to oligonucleotides, nucleotides,polynucleotides, or to a fragment of any of these, to DNA or RNA (e.g.,mRNA, rRNA, tRNA, miRNA, siRNA) of genomic or synthetic origin which maybe single-stranded or double-stranded and may represent a sense orantisense strand, to peptide nucleic acids, or to any DNA-like orRNA-like material, natural or synthetic in origin, including, e.g.,iRNA, ribonucleoproteins (e.g., iRNPs). The term can also encompassnucleic acids containing known analogues of natural nucleotides, as wellas nucleic acid-like structures with synthetic backbones.

The term “subject” can refer to any animal, including, but not limitedto, humans and non-human animals (e.g., rodents, arthropods, insects,fish (e.g., zebrafish)), non-human primates, ovines, bovines, ruminants,lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.),which is to be the recipient of a particular treatment. Typically, theterms “patient” and “subject” are used interchangeably herein inreference to a human subject.

The terms “inhibit,” “silencing,” and “attenuating” can refer to ameasurable reduction in expression of a target mRNA (or thecorresponding polypeptide or protein) as compared with the expression ofthe target mRNA (or the corresponding polypeptide or protein) in theabsence of an interfering RNA molecule of the present invention. Thereduction in expression of the target mRNA (or the correspondingpolypeptide or protein) is commonly referred to as “knock-down” and isreported relative to levels present following administration orexpression of a non-targeting control RNA.

The term “antisense” is used in reference to RNA sequences which arecomplementary to a specific RNA sequence (e.g., mRNA). Antisense RNA maybe produced by any method, including synthesis by splicing the gene(s)of interest in a reverse orientation to a viral promoter which permitsthe synthesis of a coding strand. Once introduced into a cell, thistranscribed strand combines with natural mRNA produced by the cell toform duplexes. These duplexes then block either the furthertranscription of the mRNA or its translation. In this manner, mutantphenotypes may be generated. The term “antisense strand” is used inreference to a nucleic acid strand that is complementary to the “sense”strand. The designation (−) (i.e., “negative”) is sometimes used inreference to the antisense strand, with the designation (+) sometimesused in reference to the sense (i.e., “positive”) strand.

The terms “siRNA” refers to either small interfering RNA, shortinterfering RNA, or silencing RNA. Generally, siRNA comprises a class ofdouble-stranded RNA molecules, approximately 20-25 nucleotides inlength. Most notably, siRNA is involved in RNA interference (RNAi)pathways and/or RNAi-related pathways, wherein the compounds interferewith gene expression.

The term “shRNA” refers to any small hairpin RNA or short hairpin RNA.Although it is not necessary to understand the mechanism of aninvention, it is believed that any sequence of RNA that makes a tighthairpin turn can be used to silence gene expression via RNAinterference. Typically, shRNA uses a vector stably introduced into acell genome and is constitutively expressed by a compatible promoter.The shRNA hairpin structure may also cleaved into siRNA, which may thenbecome bound to the RNA-induced silencing complex (RISC). This complexbinds to and cleaves mRNAs which match the siRNA that is bound to it.

The term “microRNA”, “miRNA”, or “.mu.RNA” refers to any single-strandedRNA molecules of approximately 21-23 nucleotides in length, whichregulate gene expression. miRNAs may be encoded by genes from whose DNAthey are transcribed but miRNAs are not translated into protein (i.e.they are non-coding RNAs). Each primary transcript (a pri-miRNA) isprocessed into a short stem-loop structure called a pre-miRNA andfinally into a functional miRNA. Mature miRNA molecules are partiallycomplementary to one or more messenger RNA (mRNA) molecules, and theirmain function is to down-regulate gene expression.

Embodiments described herein relate to compounds used to formmultifunctional pH-sensitive carriers that are designed to condensenucleic acids, such as siRNA, and deliver the condensed nucleic acids tocells. The compounds can include a protonable amino head group, fattyacid or lipid tails, and two cysteine residues.

The protonable amino head group can complex with nucleic acids to formnanoparticles for delivery of nucleic acids to cells. The amines in thehead groups contribute to the essential pH-sensitive characteristic ofthe carrier system, which is important for improving endosomal escapeand RNAi-mediated silencing efficiency. Greater protonation of the aminohead groups can occur in the relatively acidic environment (pH=5-6) ofthe endosome and lysosome compartments after cellular uptake. Thisenhances electrostatic interactions between the cationic carriers andthe anionic phospholipids of endosomal/lysosomal membranes, promotingthe bilayer destabilization and nanoparticle charge neutralizationevents required for efficient cytosolic release of their nucleic acidpayload. By affecting the number of amines, and thus overall pKa, of thecationic carrier, the choice of head group can ultimately determine thedegree to which such protonation can occur. The pH-sensitive property ofthe carrier system is essential so that the nanoparticles do not affectthe integrity of the outer cell membrane and cause cell death, butinstead are able to selectively fuse with and destabilize the endosomaland lysosomal membranes.

The cysteine residues can form disulfide bridges via autooxidation andreact with functional groups of other compounds, such as thosecontaining thiol groups. Once the nucleic acid is complexed with thecompound, the thiol groups can produce disulfide (S—S) bonds or bridgesby autooxidation to form oligomers and polymers or cross-linking. Thedisulfide bonds can stabilize the nanoparticles of the nucleic acid andcompound and help achieve release of the nucleic acid once thenanoparticle is in the cell.

For example, when the nucleic acid is siRNA, the cleavage of disulfidebonds in the siRNA delivery system in reductive cytoplasm can facilitatecytoplasm-specific release of siRNA. The compounds will be stable in theplasma at very low free thiol concentration (e.g., 15 μM). When thecompounds are incorporated into target cells, the high concentration ofthiols present in the cell (e.g., cytoplasm) will reduce the disulfidebonds to facilitate the dissociation and release of the nucleic acid.

The disulfide bonds can be readily produced by reacting the same ordifferent compounds before complexation with nucleic acid or during thecomplexation in the presence of an oxidant. The oxidant can be air,oxygen or other chemical oxidants. Depending upon the dithiol compoundselected and oxidative conditions, the degree of disulfide formation canvary in free polymers or in complexes with nucleic acids. Thus, thecompounds including two cysteine residues are monomers, and the monomerscan be dimerized, oligomerized, or polymerized depending upon thereaction conditions.

The fatty acid or lipid tails groups can participate in hydrophobiccondensation and help form compact, stable nanoparticles with thenucleic acids and introduce amphiphilic properties to facilitate pHsensitive escape of nanoparticles from endosomal and lysosomalcompartments. This is particularly useful when the compounds are used asin vivo delivery devices.

In general, the transfection efficiency of carriers has been shown todecrease with increasing alkyl chain length and saturation of the lipidtail groups. When saturated, shorter aliphatic chains (C12 and C14)favor higher rates of inter-membrane lipid mixing and reportedly allowfor better transfection efficiencies in vitro, as compared to in vivo,whereas the opposite is true for longer chains (C16 and C18). Typically,saturated fatty acids greater than 14 carbons in length are notfavorable for nucleic acid transfections due to their elevated phasetransition temperature and overall less fluidity than those that areunsaturated. However, it has been discovered that there exists a limit,at which point an increase in unsaturation and lipid fluidity isinversely correlated to transfection efficiency, primarily because somedegree of rigidity is required for particle stability, as evidenced bythe widespread use of cholesterol in lipid nanoparticle formulations.

Advantageously, multifunctional pH-sensitive carriers formed using thecompounds have improved stability when administered systemically to asubject, protect condensed nucleic acids from degradation, and promoteendosomal escape and cytosolic release upon cellular uptake.

In some embodiments, a targeting group can be attached to the compoundby, for example, a thiol group of a cysteine residue. The targetinggroup can be useful in the delivery of nucleic acids into cells. Thetargeting group can be a peptide, an antibody, an antibody fragment orone of their derivatives. For example, target-specific peptides can beconjugated directly to the compound or indirectly via a linker (e.g.,polyethylene glycol) prior or during the formation of nanoparticles.Depending upon the selection of the targeting group, the targeting groupcan be covalently bonded to either the thiol group of the cysteineresidues.

In one aspect, the targeting group is indirectly attached to thecompound by a linker. Examples of linkers include, but are not limitedto, a polyamine group, a polyalkylene group, a polyamino acid group or apolyethylene glycol group. The selection of the linker as well as themolecular weight of the linker can vary depending upon the desiredproperties. In one aspect, the linker is polyethylene glycol having amolecular weight from 500 to 10,000, 500 to 9,000, 500 to 8,000, 500 to7,000, or 2,000 to 5,000. In certain aspects, the targeting group isfirst reacted with the linker in a manner such that the targeting groupis covalently attached to the linker. For example, the linker canpossess one or more groups that can react with an amino group present ona peptide. The linker also possesses additional groups that react withand form covalent bonds with the compounds described herein. Forexample, the linker can possess maleimide groups that readily react withthe thiol groups. The selection of functional groups present on thelinker can vary depending upon the functional groups present on thecompound. In one aspect, the targeting group is a peptide such as, anRGD peptide or bombesin peptide that is covalently attached topolyethylene glycol.

In some embodiments, the linker can include an acid labide bond, such asformed by incorporation of a hydazone into the linker, that ishydrolyzable in an endolysomal environment following uptake to cells,such as cancer cells. For example, the linker can be covalently linkedto the compound by at least one of a covalent hydrolyzable ester,covalent hydrolyzable amide, covalent photodegradable urethane, orcovalent hydrolyzable acrylate-thiol linkage. Following cellular uptakeof the compound, within the endosomes, the increasingly acidicenvironment can cleave the acid labile linkage to promote shedding of apolymer linker, such as PEG, and expose the core of the compound/nucleiccomplex nanoparticle.

In other aspects, it is also desirable to attach the targeting group toa nanoparticle produced by the compounds described herein. For example,after a nanoparticle composed of a nucleic acid has been produced usingthe compounds and techniques described herein, the targeting group canbe attached to the nanoparticle via a linker.

In some embodiments, the compound can include formula (I):

-   -   wherein R¹ is an akylamino group or a group containing at least        one aromatic group; R² and R³ are independently an aliphatic        group or a hydrophobic group, derived, for example, from a fatty        acid;    -   R⁴ and R⁵ are independently H, a substituted or unsubstituted        akyl group, an akenyl group, an acyl group, an aromatic group,        polymer, a targeting group, or a detectable moiety; a, b, c, and        d are independently an integer from 1 to 10 (e.g., a, b, c, and        d are each 2); and pharmaceutically acceptable salts thereof.

In some embodiments, R¹ can include at least one of:

-   -   where R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are        independently hydrogen, an alkyl group, a hydrophobic group, or        a nitrogen containing substituent; and e, f, g, i, j, k, and 1,        are an integer from 1 to 10.

For example, R¹ can include at least one of CH₂NH₂, CH₂CH₂NH₂,CH₂CH₂CH₂NH₂, CH₂CH₂CH₂CH₂NH₂, CH₂CH₂CH₂CH₂CH₂NH₂, CH₂NHCH₂CH₂CH₂NH₂,CH₂CH₂NHCH₂CH₂CH₂NH₂, CH₂CH₂CH₂NHCH₂CH₂CH₂CH₂NHCH₂CH₂CH₂NH₂,CH₂CH₂NHCH₂CH₂CH₂CH₂NH₂, CH₂CH₂NHCH₂CH₂CH₂NHCH₂CH₂CH₂HN₂, orCH₂CH₂NH(CH₂CH₂NH)_(d)CH₂CH₂NH₂, where d is from 0 to 10.

In some embodiments, R¹ can be CH₂CH₂NH₂ orCH₂CH₂NHCH₂CH₂CH₂NHCH₂CH₂CH₂HN₂. In other embodiments, R¹ is CH₂CH₂NH₂.

In other embodiments, R² and R³ are independently an aliphatic group ora hydrophobic group derived from fatty acid, such as oleic acid orlinoleic acid, and are the same or different. The additional double bondin linoleic acid introduces an extra kink into the hydrocarbon backbone,giving the compound a broader conical shape than oleic acid andincreasing its fluidity. When incorporated into a nanoparticlestructure, the extra degree of unsaturation elevates the propensity toform the hexagonal phase during an impending membrane fusion event ofcellular uptake.

In some embodiments, R⁴ and R⁵ are independently H, a substituted orunsubstituted polymer, a targeting group, or a detectable moiety.

In some embodiments, the compound can have the formulas:

-   -   or pharmaceutically acceptable salts thereof.

The compounds having the general formula I can be synthesized usingsolid phase techniques known in the art. FIG. 1 illustrates an exampleof a synthetic procedure for preparing the compounds. In general, theapproach in FIG. 1 involves the systematicprotection/elongation/deprotection to produce a dithiol compound. Thehydrophobic group is produced by reacting oleic acid with the aminogroup present on the cysteine residue. Although FIG. 1 depicts oneapproach for producing the compounds of formula I, other synthetictechniques can be used.

Any of the compounds described herein can exist or be converted to thesalt thereof. In one aspect, the salt is a pharmaceutically acceptablesalt. The salts can be prepared by treating the free acid with anappropriate amount of a chemically or pharmaceutically acceptable base.Representative chemically or pharmaceutically acceptable bases areammonium hydroxide, sodium hydroxide, potassium hydroxide, lithiumhydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide,zinc hydroxide, copper hydroxide, aluminum hydroxide, ferric hydroxide,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, lysine, arginine, histidine, and the like. In oneaspect, the reaction is conducted in water, alone or in combination withan inert, water-miscible organic solvent, at a temperature of from about0° C. to about 100° C., such as at room temperature. The molar ratio ofthe compound to base used is chosen to provide the ratio desired for anyparticular salts. For preparing, for example, the ammonium salts of thefree acid starting material, the starting material can be treated withapproximately one equivalent of base to yield a salt.

In another aspect, any of the compounds described herein can exist or beconverted to the salt with a Lewis base thereof. The compounds can betreated with an appropriate amount of Lewis base. Representative Lewisbases are ammonium hydroxide, sodium hydroxide, potassium hydroxide,lithium hydroxide, calcium hydroxide, magnesium hydroxide, ferroushydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide, ferrichydroxide, isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, lysine, arginine, histidine, THF, ether, thiolreagent, alcohols, thiol ethers, carboxylates, phenolates, alkoxides,water, and the like. In one aspect, the reaction is conducted in water,alone or in combination with an inert, water-miscible organic solvent,at a temperature of from about 0° C. to about 100° C. such as at roomtemperature. The molar ratio of the compound to base used is chosen toprovide the ratio desired for any particular complexes. For example, theammonium salts of the free acid starting material, the starting materialcan be treated with approximately one equivalent of chemically orpharmaceutically acceptable Lewis base to yield a complex.

If the compounds possess carboxylic acid groups, these groups can beconverted to pharmaceutically acceptable esters or amides usingtechniques known in the art. Alternatively, if an ester is present onthe dendrimer, the ester can be converted to a pharmaceuticallyacceptable ester using transesterification techniques.

The compounds described herein have numerous applications with respectto the delivery of nucleic acids to a subject. In some embodiments, thecompounds described herein can be used in gene therapy to delivernucleic acid or genetic materials to cells and tissues.

The nucleic acid can be an oligonucleotide, deoxyribonucleic acid (DNA),ribonucleic acid (RNA), or peptide nucleic acid (PNA). The nucleic acidof interest introduced by the present method can be nucleic acid fromany source, such as a nucleic acid obtained from cells in which itoccurs in nature, recombinantly produced nucleic acid, or chemicallysynthesized nucleic acid. For example, the nucleic acid can be cDNA orgenomic DNA or DNA synthesized to have the nucleotide sequencecorresponding to that of naturally-occurring DNA. The nucleic acid canalso be a mutated or altered form of nucleic acid (e.g., DNA thatdiffers from a naturally occurring DNA by an alteration, deletion,substitution or addition of at least one nucleic acid residue) ornucleic acid that does not occur in nature.

In one aspect, the nucleic acid can be a functional nucleic acid.Functional nucleic acids are nucleic acid molecules that have a specificfunction, such as binding a target molecule or catalyzing a specificreaction. Functional nucleic acid molecules can be divided into thefollowing categories, which are not meant to be limiting. For example,functional nucleic acids include antisense molecules, aptamers,ribozymes, triplex forming molecules, siRNA, miRNA, shRNA, IncRNA andexternal guide sequences. The functional nucleic acid molecules can actas affectors, inhibitors, modulators, and stimulators of a specificactivity possessed by a target molecule, or the functional nucleic acidmolecules can possess a de novo activity independent of any othermolecules.

Functional nucleic acids can be a small gene fragment that encodesdominant-acting synthetic genetic elements (SGEs), e.g., molecules thatinterfere with the function of genes from which they are derived(antagonists) or that are dominant constitutively active fragments(agonists) of such genes. SGEs can include, but are not limited to,polypeptides, inhibitory antisense RNA molecules, ribozymes, nucleicacid decoys, and small peptides. The small gene fragments and SGElibraries disclosed in U.S. Patent Publication No. 2003/0228601, whichis incorporated by reference, can be used herein.

The functional nucleic acids of the present method can function toinhibit the function of an endogenous gene at the level of nucleicacids, e.g., by an antisense, RNAi or decoy mechanism. Alternatively,certain functional nucleic acids can function to potentiate (includingmimicking) the function of an endogenous gene by encoding a polypeptidethat retains at least a portion of the bioactivity of the correspondingendogenous gene, and may in particular instances be constitutivelyactive.

Other therapeutically important nucleic acids include antisensepolynucleotide sequences useful in eliminating or reducing theproduction of a gene product, as described by Tso, P. et al Annals NewYork Acad. Sci. 570:220-241 (1987). Also contemplated is the delivery ofribozymes. These antisense nucleic acids or ribozymes can be expressed(replicated) in the transfected cells. Therapeutic nucleic acids orpolynucleotides useful herein can also code for immunity-conferringpolypeptides, which can act as endogenous immunogens to provoke ahumoral or cellular response, or both. The nucleic acids orpolynucleotides employed can also code for an antibody. In this regard,the term “antibody” encompasses whole immunoglobulin of any class,chimeric antibodies and hybrid antibodies with dual or multiple antigenor epitope specificities, and fragments, such as F(ab)₂, Fab², Fab andthe like, including hybrid fragments. Also included within the meaningof “antibody” are conjugates of such fragments, and so-called antigenbinding proteins (single chain antibodies) as described, for example, inU.S. Pat. No. 4,704,692, the contents of which are hereby incorporatedby reference.

In some embodiments, the nucleic acid is siRNA. siRNAs are doublestranded RNA molecules (dsRNAs) with approximately 20 to 25 nucleotides,which are generated by the cytoplasmic cleavage of long RNA with theRNase III enzyme Dicer. siRNAs specifically incorporate into theRNA-induced silencing complex (RISC) and then guide the RNAi machineryto destroy the target mRNA containing the complementary sequences. SinceRNAi is based on nucleotide base-pairing interactions, it can betailored to target any gene of interest, rendering siRNA an ideal toolfor treating diseases with gene silencing. Gene silencing with siRNAshas a great potential for the treatment of human diseases as a newtherapeutic modality. Numerous siRNAs have been designed and reportedfor various therapeutic purposes and some of the siRNAs havedemonstrated specific and effective silencing of genes related to humandiseases. Therapeutic applications of siRNAs include, but are notlimited to, inhibition of viral gene expression and replication inantiviral therapy, anti-angiogenic therapy of ocular diseases, treatmentof autoimmune diseases and neurological disorders, and anticancertherapy. Therapeutic gene silencing has been demonstrated in mammals,which bodes well for the clinical application of siRNA. It is believedthat siRNA can target every gene in human genome and has unlimitedpotential to treat human disease with RNAi.

In some embodiments, the nucleic acid can be an RNAi nucleic acid, suchas siRNA, that inhibits the expression of long noncoding RNA (lncRNA),such as onco-lncRNA. LncRNAs are the largest class of noncoding RNAs,comprising over 20,000 genes annotated in the ENcylopedia of DNAElements (ENCODE) and other reference databases. They are defined astranscripts produced by RNA polymerase II that are longer than 200nucleotides and devoid of an open reading frame that can be translatedinto a protein. Derrien et al., 2012. “The GENCODE v7 catalog of humanlong noncoding RNAs: analysis of their gene structure, evolution, andexpression. Genome Res 22: 1775-1789.

The function of most lncRNAs is unknown, but they have been proposed toplay roles in both negatively and positively regulating gene expression.They can regulate expression of protein-coding genes at both thetranscriptional and posttranscriptional levels. Posttranscriptionalregulation could occur by competing with endogenous RNA to regulatemicroRNA levels, modulating mRNA stability and translation by homologousbase pairing, or altering cellular localization of mRNAs.Transcriptional regulation can occur in cis with their effectsrestricted to the chromosome from which they are transcribed and intrans with their effects targeting gene transcription on otherchromosomes. Both cis- and trans-acting lncRNAs can mediate theireffects through their RNA transcripts; cis-acting lncRNAs can alsoregulate gene transcription as a result of the process of splicing or oftranscription itself.

LncRNAs have been implicated in the regulation of tissue anddevelopmental stage-specific transcription of genes. Mutations anddysregulation of lncRNAs have been increasingly linked with diversehuman diseases. Wapinski et al., 2011. “Long noncoding RNAs and humandisease” Trends Cell Biol 21: 354-361. Despite their pervasivetranscription, very little is currently known about the regulation andfunction of lncRNAs. They have been proposed to play roles in bothnegatively and positively regulating gene expression. Their functionsare determined by their secondary and tertiary structures and they donot share sequence homology with their targets. Recent reports havedemonstrated a role for lncRNA in the regulation of Toll-like-receptormediated immune responses and differentiation and function of humandendritic cells and T cells, implicating them in immune mechanisms thatcould modulate autoimmune disease. Mauger et al., 2013. “The geneticcode as expressed through relationships between mRNA structure andprotein function” FEBS Lett 587: 1180-1188; Carpenter et al., 2013. “Along noncoding RNA mediates both activation and repression of immuneresponse genes” Science 341:789-792; Wang et al., 2014. “TheSTAT3-binding long noncoding RNA Inc-DC controls human dendritic celldifferentiation” Science 344: 310-313; Hu et al., 2013. “Expression andregulation of intergenic long noncoding RNAs during T cell developmentand differentiation” Nat Immunol 14: 1190-1198; Spurlock et al., 2015.“Expression and functions of long noncoding RNAs during human T helpercell differentiation” Nat Commun 6: 6932; and Collier et al., 2012.“Cutting edge: influence of Tmevpg1, a long intergenic noncoding RNA, onthe expression of lfng by Th1 cells” J Immunol 189: 2084-2088.

In some embodiments, the nucleic acid can be an RNAi nucleic acid, suchas siRNA, that inhibits the expression of onco-lncRNA. LncRNAs haverecently been identified in the development of multiple oncogenicprocesses, including EMT, inflammation, cancer sternness, metastasis,and drug-resistance. Aberrant overexpression of oncogenic IncRNAs(onco-lncRNAs) is disease- and tissue-specific, and can influence globalgene signatures and clinical outcomes by regulating multi-level geneexpression. Examples of onco-lncRNAs include DANCR, NBAT1, LINC00511,LINC-UBC-1, ANCR, BCAR4, TINCR, BANCR, HOTTP, HOTAIR, CCAT1, HULC,LCAL1, MEG3, UCA1, FENDRR, and ANRIL. SiRNA that inhibits expression ofonco-lncRNAs can be complexed with the carrier compounds describedherein and administered to a subject with cancer to inhibit expressionof the onco-lncRNAs and treat cancer in the subject. Examples of siRNAthat inhibit the expression of onco-lncRNA described herein can includesiDANCR, siBANCR, and siHOTTP. Other examples of siRNA that inhibit theexpression of onco-lncRNA and that can be complexed with carriercompounds described herein can be used in treating cancer in a subject.

The nucleic acid can be complexed to the carrier compounds describedherein by admixing the nucleic acid and the compound or correspondingdisulfide oligomer or polymer. The pH of the reaction can be modified toconvert the amino groups present on the compounds described herein tocationic groups. For example, the pH can be adjusted to protonate theamino group. With the presence of cationic groups on the compound, thenucleic acid can electrostatically bond (i.e., complex) to the compound.In one aspect, the pH is from 1 to 7.4.

In another aspect, the N/P ratio of the carrier compounds complexed withnucleic acids can be from 0.5 to 100, where N is the number of nitrogenatoms (e.g., amines) present on the compounds that can form a positivecharge and P is the number of phosphate groups present on the nucleicacids. Thus, by modifying the compound with the appropriate number ofamino groups in the head group, it is possible to tailor the bonding(e.g., type and strength of bond) between the nucleic acid and thecompound. The N/P ratio can be adjusted depending on the cell type towhich the nucleic acid is to be delivered. In some embodiments where thecell is cancer, the N/P ratio can be at least about 6, at least about10, or at least about 15. In other embodiments, the N/P ration can befrom about 6 to about 20, about 10 to about 20, about 12 to about 20, orabout 6 about 14.

In one aspect, the nucleic acid/carrier complex is a nanoparticle. Inone aspect, the nanoparticle has a diameter of about 1000 nanometers orless, for example, about 50 nm to about 200 nm, about 60 nm to about 180nm, about 70 nm to about 160 nm, about 80 nm to about 140 nm, or about90 nm to about 120 nm.

In other aspects, the compounds described herein can be designed so thatthe resulting nucleic acid nanoparticle escapes endosomal and/orlysosomal compartments at the endosomal-lysosomal pH. For example, thecompound forming nanoparticles with nucleic acids can be designed suchthat its structure and amphiphilicity changes at endosomal-lysosomal pH(5.0-6.0) and disrupts endosomal-lysosomal membranes, which allows entryof the nanoparticle into the cytoplasm. In one aspect, the ability ofspecific endosomal-lysosomal membrane disruption of the compoundsdescribed herein can be tuned by modifying their pH sensitiveamphiphlicity by altering the number and structure of protonatableamines and lipophilic groups. For example, decreasing the number ofprotonatable amino groups can reduce the amphiphilicity of ananoparticle produced by the compound at neutral pH. In one aspect, thecompounds herein have 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to8, 1 to 6, 1 to 4, or 2 protonatable amino or substituted amino groups.The pH-sensitive amphiphilicity of the compounds and nanoparticlesproduced by the compounds can be used to fine-tune the overall pKa ofthe nanoparticle. Low amphiphilicity of the nanoparticles atphysiological pH can minimize non-specific cell membrane disruption andnonspecific tissue uptake of the nucleic acid. In certain aspects, it isdesirable that the carriers have low amphiphilicity at the physiologicalpH and high amphiphilicity at the endosomal-lysosomal pH, which willonly cause selective endosomal-lysosomal membrane disruption with thenanoparticles.

The surface of the nanoparticle complexes can be modified by, forexample, covalently incorporating polyethylene glycol by reactingunpolymerized free thiol of the nanoparticle to reduce non-specifictissue uptake in vivo. For example, PEG-maleimide reacts rapidly withfree thiol groups. The molecular weight of the PEG can vary dependingupon the desired amount of hydrophilicity to be imparted on the carrier.PEG-modification of the carrier can also protect nanoparticles composedof the nucleic acid from enzymatic degradation upon uptake by the cell(e.g., endonucleases). Targeting groups, including peptides, proteins,antibodies or antibody fragment, can also be incorporated into thenanoparticle complexes during the preparation of the complexes toenhance the delivery specificity and efficiency of the genetic materialsto the target cells. Polyethylene glycol can be used as the spacer toconjugate targeting agents to the nanoparticle complexes.

The compounds described herein can be used to introduce a nucleic acidinto a cell. The method generally involves contacting the cell with acomplex, wherein the nucleic acid is taken up into the cell. In oneaspect, the compounds described herein can facilitate the delivery ofDNA or RNA as therapy for genetic disease by supplying deficient orabsent gene products to treat any genetic disease or by silencing geneexpression. Techniques known in the art can used to measure theefficiency of the compounds described herein to deliver nucleic acids toa cell.

The term “cell” as used herein is intended to refer towell-characterized homogenous, biologically pure populations of cells.These cells may be eukaryotic cells that are neoplastic or which havebeen “immortalized” in vitro by methods known in the art, as well asprimary cells, or prokaryotic cells. The cell line or host cell ispreferably of mammalian origin, but cell lines or host cells ofnon-mammalian origin may be employed, including plant, insect, yeast,fungal or bacterial sources.

In one aspect, the cell comprises stem cells, committed stem cells,differentiated cells, primary cells, and tumor cells. Examples of stemcells include, but are not limited to, embryonic stem cells, bone marrowstem cells and umbilical cord stem cells. Other examples of cells usedin various embodiments include, but are not limited to, osteoblasts,myoblasts, neuroblasts, fibroblasts, glioblasts, germ cells,hepatocytes, chondrocytes, keratinocytes, smooth muscle cells, cardiacmuscle cells, connective tissue cells, glial cells, epithelial cells,endothelial cells, hormone-secreting cells, cells of the immune system,and neurons.

Atypical or abnormal cells, such as tumor cells, can also be usedherein. Tumor cells cultured on substrates described herein can providemore accurate representations of the native tumor environment in thebody for the assessment of drug treatments. Growth of tumor cells on thesubstrates described herein can facilitate characterization ofbiochemical pathways and activities of the tumor, including geneexpression, receptor expression, and polypeptide production, in an invivo-like environment allowing for the development of drugs thatspecifically target the tumor.

The complexes (i.e., nanoparticles) described above can be administeredto a subject using techniques known in the art. For example,pharmaceutical compositions can be prepared with the complexes. It willbe appreciated that the actual preferred amounts of the complex in aspecified case will vary according to the specific compound beingutilized, the particular compositions formulated, the mode ofapplication, and the particular sites and subject being treated. Dosagesfor a given host can be determined using conventional considerations,e.g., by customary comparison of the differential activities of thesubject compounds and of a known agent, e.g., by means of an appropriateconventional pharmacological protocol. Physicians and formulators,skilled in the art of determining doses of pharmaceutical compounds,will have no problems determining dose according to standardrecommendations (Physicians Desk Reference, Barnhart Publishing (1999).

Pharmaceutical compositions described herein can be formulated in anyexcipient the biological system or entity can tolerate. Examples of suchexcipients include, but are not limited to, water, saline, Ringer'ssolution, dextrose solution, Hank's solution, and other aqueousphysiologically balanced salt solutions. Nonaqueous vehicles, such asfixed oils, vegetable oils such as olive oil and sesame oil,triglycerides, propylene glycol, polyethylene glycol, and injectableorganic esters such as ethyl oleate can also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosol, cresols, formalin and benzyl alcohol.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration to humans,including solutions, such as sterile water, saline, and bufferedsolutions at physiological pH.

Molecules intended for pharmaceutical delivery can be formulated in apharmaceutical composition. Pharmaceutical compositions can includecarriers, thickeners, diluents, buffers, preservatives, surface activeagents and the like in addition to the molecule of choice.Pharmaceutical compositions can also include one or more activeingredients, such as antimicrobial agents, antiinflammatory agents,anesthetics, and the like.

The pharmaceutical composition can be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration can be topically, includingophthalmically, vaginally, rectally, intranasally. Administration canalso be intravenously or intraperitoneally. In the case of contactingcells with the nanoparticlar complexes of nucleic acid described herein,it is possible to contact the cells in vivo or ex vivo.

Preparations for administration include sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles, if needed forcollateral use of the disclosed compositions and methods, include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles, if needed forcollateral use of the disclosed compositions and methods, include fluidand nutrient replenishers, electrolyte replenishers (such as those basedon Ringer's dextrose), and the like. Preservatives and other additivescan also be present such as, for example, antimicrobials, anti-oxidants,chelating agents, and inert gases and the like.

Formulations for topical administration can include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like can be necessary or desirable.

Dosing is dependent on severity and responsiveness of the condition tobe treated, but will normally be one or more doses per day, with courseof treatment lasting from several days to several months or until one ofordinary skill in the art determines the delivery should cease. Personsof ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. It is understood that any givenparticular aspect of the disclosed compositions and methods can beeasily compared to the specific examples and embodiments disclosedherein, including the non-polysaccharide based reagents discussed in theExamples. By performing such a comparison, the relative efficacy of eachparticular embodiment can be easily determined. Particularly preferredcompositions and methods are disclosed in the Examples herein, and it isunderstood that these compositions and methods, while not necessarilylimiting, can be performed with any of the compositions and methodsdisclosed herein.

The following example is for the purpose of illustration only and is notintended to limit the scope of the claims, which are appended hereto.

Example 1

RNAi-based therapeutic regimens hold tremendous potential fordownregulating oncogene expression. However, clinical applications ofRNAi have been limited by challenges associated with delivering siRNA,including potential immunogenicity, poor cellular uptake of unmodifiedsiRNA, and degradation by serum nucleases. Nanoparticle-mediateddelivery of siRNA is desirable due to its ability to protect siRNAs andfacilitate their uptake into target cells. Although various deliverysystems are reported to exhibit efficient transfection, concernsregarding their clinical safety and efficacy continue to prevent theadvancement of RNAi-based therapeutics. Cationic lipid-based carriershave demonstrated remarkable delivery potential due to their ability toreadily form complexes with negatively charged siRNAs, and toefficiently promote cellular uptake of the siRNA cargo. We developed amultifunctional cationic lipid-based carrier,(1-aminoethyl)iminobis[N-oleicylsteinyl-1-aminoethyl)propionamide](ECO), which facilitates effective siRNA-mediated RNAi in various cancercell lines. ECO ionically complexes with siRNA and forms stablenanoparticles to protect the siRNA cargo from degradation (FIG. 2A).Once internalized and trafficked to the late endosomes, the pH-sensitiveamphiphilicity of ECO promotes endo-lysosomal escape of thenanoparticles, and reduction of the disulfide linkages created duringnanoparticle formation by cytosolic glutathione releases the siRNA toachieve potent gene silencing. ECO is also designed to facilitate facileand versatile functionalization of the surface of these nanoparticleswith targeting moieties.

Considering the roles of β3 integrin in tumor progression and the urgentneed for targeted therapies tailored specifically to TNBC, we sought toalleviate TNBC metastasis by silencing β3 integrin using ECO/siRNAnanoparticles. The present study demonstrates the efficacy of ECO/siβ3nanoparticles in silencing β3 integrin expression and the consequentinhibition of TGF-β-mediated EMT and invasion of breast cancer cells invitro. The nanoparticles were modified with RGD peptides via PEG spacersto improve biocompatibility and systemic target-specific delivery of thetherapeutic siβ3 in vivo. The efficacy of the RGD-targeted ECO/siβ3nanoparticles in alleviating primary and metastatic tumor burden wasdetermined in tumor-bearing mice following multiple intravenousinjections.

Preparation of ECO/siRNA Nanoparticles

ECO/siRNA nanoparticles were prepared at an N/P ratio of 8. ECO andsiRNA were diluted in equal volumes in nuclease-free water from stocksolutions of 2.5 mM in ethanol and 18.8 μM in nuclease-free water,respectively. The equal volumes of ECO and siRNA were mixed followed bya 30-min incubation period at room temperature under gentle agitation.For RGD- and RAD-modified ECO/siRNA nanoparticles, RGD-PEG-Mal orRAD-PEG-Mal (MW=3,400 Da; NANOCS, New York, N.Y.) was added into an ECOsolution at 2.5 mol % for 30 min under gentle agitation and subsequentlymixed with an equal volume of siRNA in RNase-free water for anadditional 30 min. After the incubation, free peptide derivative wasremoved from RGD- and RAD-modified ECO/siRNA nanoparticles byultrafiltration (Nanosep, MWCO=100 K, 5000×g, 5 min; Sigma Aldrich, St.Louis, Mo.). The following siRNAs were purchased from Integrated DNATechnologies (Coralville, Iowa): Mouse integrin β3 sense:[GCUCAUCUGGAAGCUACUCAUCACT] (SEQ ID NO: 1), Mouse integrin β3 antisense:[AGUGAUGAGUAGCUUCCAGAUGAGCUC] (SEQ ID NO: 2), Human integrin β3 sense:[GCUCAUCUGGAAACUCCUCAUCACC] (SEQ ID NO: 3), and Human integrin β3antisense: [GGUGAUGAGGAGUUUCCAGAUGAGCUC] (SEQ ID NO: 4).

Cell Lines and Reagents

MDA-MB-231 cells were obtained from ATCC (Manassas, Va.) and cultured inDulbecco's Modified Eagle Medium (DMEM; Gibco, Grand Island, N.Y.)supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island,N.Y.). NME were engineered as previously described³⁷ and cultured inDMEM supplemented with 10% FBS and 10 μg/mL of insulin. Both cell lineswere engineered to stably express firefly luciferase by transfectionwith pNifty-CMV-luciferase and selection with Zeocin (500 μg/ml;Invitrogen, Carlsbad, Calif.).

Western Blot Analyses

Immunoblotting analyses were performed as previously described. Briefly,NME and MDA-MB-231 cells were seeded into 6-well plates (1.5×10⁵cells/well) and allowed to adhere overnight. The cells were thenincubated in the absence or presence of TGF-β1 (5 ng/mL) for 3 d andthen treated with ECO/siRNA complexes for 4 h in complete growth medium.At each indicated time point, detergent-solubilized whole cell extracts(WCE) were prepared by lysing the cells in Buffer H (50 mMβ-glycerophosphate, 1.5 mM EGTA, 1 mM DTT, 0.2 mM sodium orthovanadate,1 mM benzamidine, 10 mg/mL leupeptin, and 10 mg/mL aprotinin, pH 7.3).The clarified WCE (20 mg/lane) were separated through 10% SDS-PAGE,transferred electrophoretically to nitrocellulose membranes, andimmunoblotted with the primary antibodies, anti-β3 integrin (1:1000;Cell Signaling) and anti-β-actin (1:1000; Santa Cruz Biotechnology).

Flow Cytometry for Nanoparticle Cellular Uptake

Cellular uptake and intracellular delivery of ECO/siRNA andRGD-ECO/siRNA nanoparticles was evaluated quantitatively using flowcytometry. ECO/siRNA and RGD-ECO/siRNA nanoparticles were prepared with40 nM Alexa Fluor 488-labeled siRNA (Qiagen; Valencia, Calif.).Approximately 2.5×10⁴ U87 cells were seeded onto 12-well plates andgrown for an additional 24 h. The cells were transfected with ECO/siRNAnanoparticles in 10% serum media. After 4 h, the transfection media wasremoved and each well was washed twice with PBS. The cells wereharvested by treatment with 0.25% trypsin containing 0.26 mM EDTA(Invitrogen; Carlsbad, Calif.), collected by centrifugation at 1,000 rpmfor 5 min, resuspended in 500 μL of PBS containing 5% paraformaldehyde,and finally passed through a 35 m cell strainer (BD Biosciences; SanJose, Calif.). Cellular internalization of the nanoparticles wasquantified by the fluorescence intensity measurement of Alexa Fluor 488for a total of 1×10⁴ cells per sample using a BD FACSCalibur flowcytometer. All the experiments were performed in triplicate and the datarepresent mean fluorescence intensity and standard deviation.

Semi-Quantitative Real-Time PCR Analyses

Real-time PCR studies were performed as described previously. Briefly,NME or MDA-MB-231 cells (100,000 cells/well) were seeded overnight onto6-well plates and treated with TGF-β (5 ng/mL) for 3 days upon deliveryof ECO nanoparticles with a non-specific siRNA or β3 integrin-specificsiRNA. At each indicated timepoint, total RNA was isolated using theRNeasy Plus Kit (Qiagen, Valencia, Calif.) and reverse transcribed usingthe iScript cDNA Synthesis System (Bio-Rad, Hercules, Calif.).Semi-quantitative real-time PCR was conducted using iQ-SYBR Green(Bio-Rad) according to manufacturer's recommendations. In all cases,differences in RNA expression for each individual gene were normalizedto their corresponding GAPDH RNA signals.

Invasion and Proliferation Assays

Invasion assays were conducted as described previously. Briefly, NMEcells, unstimulated (Pre-) or stimulated with TGF-β for 3 d (Post-EMT),were treated with the ECO/siRNA complexes for an additional 2 d. Thecells were then trypsinized and their ability to invade reconstitutedbasement membranes (5×10⁴ cells/well) was measured utilizing modifiedBoyden chambers, as previously described. For the proliferation assay,the NME cells were cultured (1×10⁴ cells/well) in the presence(post-EMT) or absence (pre-EMT) of TGF-β (5 ng/mL) for 3 d and thentreated with ECO/siRNA. Cell proliferation was determined by³H-thymidine incorporation as previously described.

3-Dimensional (3D)-organotypic cultures

3D-organotypic cultures utilizing the “on-top” method were performed asdescribed previously. NME or MDA-MB-231 cells, which were unstimulated(Pre-EMT) or stimulated with TGF-β (5 ng/mL) for 3 d (Post-EMT), werecultured in 96-well, white-walled, clear bottom tissue culture plates(2,000 cells/well) with 50 μL of Cultrex cushions (Trevigen,Gaithersburg, Md.) in media supplemented with 5% Cultrex. The cells weremaintained in culture for 4 d with continuous ECO/siRNA treatment every2 d. Growth was monitored by bright-field microscopy or bioluminescentgrowth assays (where indicated) using luciferin substrate.

Tumor Growth and Bioluminescent Imaging (BLI)

All the animal studies were performed in accordance with theInstitutional Animal Care and Use Committee for Case Western ReserveUniversity. NME cells were engineered to stably express fireflyluciferase, and were subsequently injected into the lateral tail vein offemale nude mice (1×10⁶ cells/mouse) after TGF-β stimulation (5 ng/mL)for 7 days. The pulmonary outgrowth was monitored and determined asdescribed previously. MDA-MB-231 cells, also engineered to expressfirefly luciferase and stimulated with TGF-β for 7 days, were engraftedinto the mammary fat pad of female nude mice.

Immunofluorescence and Immunohistochemical Staining

For visualization of the actin cytoskeleton, immunofluorescent analysiswas performed as previously described. NME cells (5×10⁴ cells/well) wereplated onto glass-bottom confocal dishes and allowed to adhereovernight, after which they were simultaneously stimulated with TGF-β (5ng/mL) and treated with ECO/siRNA nanoparticles, either siβ3 or siNS at100 nM siRNA concentration. After 48 and 72 h of simultaneous TGF-βstimulation and nanoparticle treatment, the cells were washed with PBS,fixed with 4% paraformaldehyde, permeabilized in 0.1% Triton-X 100,stained with Alexa Flour 488 phalloidin (25 μM; Invitrogen; Carlsbad,Calif.), and visualized under a fluorescent confocal microscope.

For immunohistochemistry, primary tumor samples were frozen in OCT,sectioned, fixed in paraffin, and maintained at −80° C. The samples werestained with H&E to evaluate the presence of tumor tissue. Forimmunofluorescence detection of fibronectin, the paraffin-embeddedslides were first deparaffinized using a series of washes in xylene anddecreasing concentrations of ethanol. Following heat-induced antigenretrieval, the samples were blocked in TBST solution containing donkeyserum and washed three times with TBST. The primary antibody (Abcam;Cambridge, Mass.) was applied for 1 h followed by three rinses withTBST. The Alexa Fluor 488 secondary antibody (Jackson; West Grove, Pa.)was applied for 1 h followed by three washes with TBST andcounterstained with DAPI. After washing with TBST and mounting in ananti-fade mounting solution (Molecular Probes), the samples were imagedusing a confocal microscope.

Results

ECO/Siβ3 Nanoparticles Induce Sustained Silencing of β Integrin

We examined the ability of ECO/β3 integrin-specific siRNA nanoparticles(ECO/siβ3) to silence β3 integrin expression in mouse NMuMG-EGFR (NME)breast cancer cells, reminiscent of a basal-like breast cancer cellline, and human MDA-MB-231 breast cancer cells, a mesenchymal-like TNBCcell line. The expression of β3 integrin was elevated in both cell linesafter stimulation with TGF-β for 72 h. Subsequent treatment of thestimulated cells with ECO/siβ3 nanoparticles resulted in the rapid lossof β3 integrin mRNA within the first 16 h following treatment (FIG. 2B).β3 integrin expression was reduced by ˜75% and this downregulation wassustained for up to 7 d in NME cells treated with TGF-β (FIGS. 1B andC). ECO/siβ3 treatment of MDA-MB-231 cells reduced β3 integrinexpression level to that of the unstimulated cells (FIGS. 2B and D).Importantly, treatment with ECO/nonspecific siRNA nanoparticles(ECO/siNS) failed to alter β3 integrin expression in both cell lines(FIGS. 2B, C, and D). Collectively, these results demonstrate theability of ECO/siβ3 nanoparticles to induce efficient and prolongedsilencing of β3 integrin expression in breast cancer cells.

ECO/Siβ3 Nanoparticles Attenuate TGF-β-Mediated EMT, Invasion, andProliferation

Next, we investigated the effects of ECO/siβ3 nanoparticles on EMT,invasion, and proliferation of breast cancer cells. Phalloidin stainingof the actin cytoskeletal architecture revealed that quiescent NME cellsdisplayed the epithelial hallmark of densely packed and well-organizedcortical actin network, while those stimulated with TGF-β exhibiteddissolved junctional complexes and acquired an elongated morphologyconsistent with stress fiber formation that are characteristic ofmesenchymal cells (FIG. 3A). Treatment of NME cells with ECO/siβ3nanoparticles at the time of TGF-β stimulation inhibited dissolution ofthe junctional complexes and stress fiber formation, while treatmentwith ECO/siNS nanoparticles failed to impact TGF-β-induced morphologicalchanges (FIG. 3A). Moreover, the phenotypic changes in post-EMT cellswere accompanied by alterations in the expression of EMT-related genes.Silencing of β3 integrin with ECO/siβ3 nanoparticles significantlyreduced TGF-β-mediated upregulation of the mesenchymal markers, N-cadand PAI-1, and inhibited TGF-β-mediated downregulation of the epithelialmarkers, E-cad and CK-19 (FIGS. 3B and C). ECO/siNS nanoparticles didnot alter the effect of TGF-β on the aforementioned EMT markers.

TGF-β-mediated EMT is also associated with increased invasiveness andcell cycle arrest. TGF-β-stimulated NME cells treated with ECO/siNSreadily invaded reconstituted basement membrane, while ECO/siβ3nanoparticles significantly inhibited invasion (FIG. 3D). Conversely,treatment of quiescent NME cells with ECO/siβ3 nanoparticles had noeffect on basal invasiveness, an event that is uncoupled from β3integrin expression. Previous studies demonstrate that parental NMuMGcells readily undergo proliferative arrest when stimulated with TGF-β.We found that EGFR overexpression in the NME cells overrides thesecytostatic effects of TGF-β (FIG. 3E), while treatment with ECO/siβ3partially restores TGF-β-mediated cytostasis (FIG. 3E). Collectively,these findings indicate that ECO/siβ3 nanoparticle-mediated silencing ofβ3 integrin attenuates TGF-β-induced EMT and invasion, and partiallyrestores TGF-β-mediated cytostasis.

ECO/Siβ3 Nanoparticles Attenuate Outgrowth of Murine and Human MECs in3D-Organotypic Culture

To study the effects of ECO/siβ3 nanoparticles in a physiologicallyrelevant system, we cultured NME and MDA-MB-231 cells in 3D-organotypiccultures to recapitulate the elastic modulus of a distant metastaticsite such as the pulmonary microenvironment. This culture methodpresented additional obstacles in the delivery and uptake ofnanoparticles, since these organoids were compact and surrounded by adense matrix. Using confocal microscopy, we confirmed that ECO/siRNAnanoparticles formulated with fluorescently-labeled siRNA (AF-488)readily gained access to NME organoids by first penetrating into theperiphery within 30 min after treatment, and further dispersingthroughout the entirety of the organoid to reach a near-uniformdistribution within 24 h (FIG. 4A). The dispersion of ECO/siRNAnanoparticles into the inner cell layers of the organoids suggests thatECO/siRNA uptake by these cells may result from diffusion throughintercellular spaces or through transcytosis. FIGS. 4B and C show thatNME and MDA-MB-231 organoids stimulated with TGF-β exhibited rapidgrowth as compared to their quiescent counterparts. Treatment withECO/siβ3 nanoparticles inhibited the growth of both quiescent andTGF-β-stimulated NME and MDA-MB-231 organoids (FIGS. 4B and C) incomparison to treatment with ECO/siNS nanoparticles. These resultsdemonstrate the effectiveness of ECO/siβ3 nanoparticles in attenuatingthe 3D outgrowth of post-EMT breast cancer cells.

Surface Modification of ECO/siRNA Nanoparticles with RGD PeptidePromotes Cellular Uptake and Sustains Gene Silencing

An essential goal of in vivo siRNA delivery is to increase siRNAlocalization at the disease site while minimizing its accumulation innon-target tissues. We modified ECO/siRNA nanoparticles with RGDpeptides via PEG spacers (3,400 Da), and examined their cellular uptakein both unstimulated and TGF-β-stimulated NME cells. TGF-β stimulationhad no effect on the cellular uptake of unmodified ECO/siRNAnanoparticles, while cellular uptake of RGD-ECO/siRNA nanoparticles wasrobustly enhanced (FIG. 5), leading to effective silencing of β3integrin in TGF-β-treated cells (FIG. 5). Since αvβ3 is a major receptorthat recognizes the RGD targeting peptide, we sought to determinewhether β3 integrin silencing impacts cellular uptake of RGD-ECO/siRNAnanoparticles. Although cellular uptake of RGD-targeted nanoparticleswas diminished upon β3 integrin silencing, uptake was nonethelesselevated consistently, because of the presence of other receptors forthe peptide (FIG. 5). Taken together, these results show thatRGD-targeted ECO/siRNA nanoparticles efficiently promote cellular uptakeand robust gene silencing, particularly in post-EMT and metastaticbreast cancer cells.

RGD-ECO/Siβ3 Nanoparticles Safely Inhibit Pulmonary Outgrowth of MouseMECs In Vivo

To address safety concerns, we examined the acute inflammatory responseof intravenously injected PEGylated ECO/siRNA nanoparticles. We examinedthe acute inflammatory response of intravenously injected PEGylatedECO/siRNA nanoparticles, to address potential safety concerns. Comparedto unmodified nanoparticles, PEGylated nanoparticles, which harbor areduced surface zeta potential, attenuated the elevation of seruminterleukin-6 (IL-6) and tumor necrotic factor-α (TNF-α) levels at 4 hpost-injection, which were resolved within 24 h. To evaluate the effectof β3 integrin silencing on pulmonary outgrowth, we inoculatedTGF-β-treated NME cells into the lateral tail vein of nude mice andsubsequently monitored pulmonary outgrowth. Systemic injections ofRGD-targeted ECO/siβ3 nanoparticles dramatically inhibited pulmonaryoutgrowth of post-EMT NME cells, as compared to non-specificRAD-ECO/siβ3 and RGD-ECO/siNS treatment groups. These resultsdemonstrate that PEGylated ECO/siRNA nanoparticles exhibit a good safetyprofile for systemic siRNA delivery, and that RGD-targeted ECO/siβ3nanoparticles with PEG spacers can effectively inhibit pulmonaryoutgrowth of TGF-β-stimulated NME cells, when targeted for in vivodelivery applications.

RGD-ECO/Siβ3 Nanoparticles Effectively Inhibit Primary Tumor Growth andMetastasis of Malignant Human MECs

To further evaluate the in vivo effect of our targeted ECO/siβ3nanoparticles, MDA-MB-231 cells pretreated with TGF-β were engraftedinto the mammary fat pad of nude mice. Mice were treated withRGD-ECO/siβ3 (1.5 mg/kg siRNA) every 5 days, starting at day 17. Primarytumor burden was monitored by bioluminescence imaging (BLI) and calipermeasurements. Compared to the untreated control, RGD-ECO/siNS orRAD-ECO/siβ3 treatment groups, RGD-ECO/siβ3 treated mice exhibitedsignificantly reduced primary tumor burden (FIG. 6). The primary tumorswere resected at week 9 (FIG. 6) and weighed. FIG. 6 shows thatRGD-ECO/siβ3 treatment resulted in significantly reduced tumor weightsas compared to the control groups. Importantly, the therapeutic efficacyof RGD-ECO/siβ3 was reflected by decreased mRNA expression of β3integrin in the primary tumors, relative to that in the control groups(FIG. 6). RAD-ECO/siβ3 treatment resulted in marginally reduced β3integrin expression (FIG. 6), which was consistent with the marginallyreduced primary tumor burden, which were not statistically significant(FIG. 6). These data reflect partial uptake of the RAD-ECO/siβ3nanoparticles by primary tumors as a result of passive tumoraccumulation attributed to tumor vascular hyperpermeability. H&Estaining of tissue sections demonstrated similar histopathologicalpatterns in RGD-ECO/siβ3-treated and control groups, while untreatedmice developed tumors that were more vascularized thanRGD-ECO/siβ3-treated tumors. Further immunostaining of tissue sectionsindicated that RGD-ECO/siβ3-treated primary tumors exhibited decreasedexpression of the mesenchymal marker, fibronectin (FIG. 6), which isassociated with poor overall survival.

RGD-ECO/siβ3 treatment resulted in robust inhibition of tumor metastasis(FIGS. 7A and C) and primary tumor recurrence (FIG. 7B), as compared tocontrol groups at week 12 post-engraftment. Interestingly, RAD-ECO/siβ3treatment also mediated significant inhibition of tumor metastasis andprimary tumor recurrence as compared to RGD-ECO/siNS treatment, but to alesser extent than RGD-ECO/siβ3. This decrease in the efficacy ofRAD-ECO/siβ3 could be attributed to the lack of specific targeting andbinding of the nanoparticles to the cancer cells. At 12 weekspost-engraftment, the RGD-ECO/siβ3 group was released from nanoparticletreatment to evaluate the lasting effects of therapeutic β3 integrinsilencing on tumor recurrence and metastasis in comparison with theuntreated control group. At 4 weeks post-treatment release (16 weekspost-engraftment), the RGD-ECO/siβ3-treated mice remained tumor-free,while the tumor burden of untreated mice continued to increase (FIGS. 7Dand E). Finally, throughout the entire course of treatment, nosignificant difference was observed in the body weights across thedifferent treatment groups, demonstrating the low toxicity of theintravenously administered, targeted PEGylated ECO/siRNA nanoparticles.Collectively, these data highlight the safety and effectiveness of thesystemic administration of RGD-ECO/siβ3 nanoparticles for the inhibitionof TNBC tumor progression and metastasis.

The data demonstrates that the RGD-ECO/siβ3 nanoparticles constitute aneffective targeted therapy to combat TNBC. Cancer metastasis involves acascade of events, including EMT and local invasion, intravasation,survival in circulation, extravasation, and outgrowth of disseminatedcells at the secondary site. In order to alleviate metastasis, it isessential to eliminate post-EMT cells and prevent their metastaticdissemination and outgrowth. Silencing EMT-related genes by RNAi has thepotential to revolutionize current treatment standards. β3 integrin hasbeen implicated as a powerful inducer of EMT, potentiating the oncogeniceffects of TGF-β by inducing invasion and metastasis of MECs. Althoughfunctional disruption of β3 integrin was shown to attenuateTGF-β-mediated EMT and tumor progression, the utilization of β3 integrinsiRNA as a therapeutic regimen was previously limited due to the lack ofa clinically feasible approach. Here, we highlight how RGD-ECO/siβ3nanoparticles inhibit TNBC metastasis by silencing the expression of β3integrin. The inhibition of TGF-β-mediated EMT with ECO/siβ3nanoparticles was evident by the obstruction of TGF-β-mediatedmorphological changes, downregulation of epithelial markers, andupregulation of mesenchymal markers. More importantly, the attenuatedEMT phenotype, decreased invasiveness resulted in reduced outgrowth ofbreast cancer cells in 3D-cultures and in vivo, as well as abrogatedmetastatic dissemination, and subsequently decreased metastatic burden.

Example 2

In this Example, we developed a dual pH-sensitive and peptide-targetedsiRNA delivery system. While various attempts have been made to createeIF4E therapies, siRNA-mediated strategies have yet to be employed invivo. Furthermore, the effect of eIF4E silencing in a drug-resistantTNBC cell line has yet to be established or be translated into a viablesystemically-administered therapeutic regimen. The inclusion of theacid-labile hydrazone bond within a peptide-targeted PEG moiety(RGD-PEG(HZ)-ECO/siRNA) created a pH-cleavable coating that sheds fromthe core ECO/siRNA nanoparticle in response to the acidic endolysosomalenvironment following uptake into tumor cells. Once the core ECO/siRNAnanoparticle has been re-exposed, the intrinsic pH-sensitiveamphiphilicity of ECO/siRNA nanoparticles enables endolysosomal membranedisruption and escape to achieve potent gene silencing. Accordingly, thepH-cleavable nanoparticles will be leveraged to silence eIF4E andresensitize drug-resistant TNBC to paclitaxel therapy.

Materials and Methods

Cell Culture

Human triple-negative breast cancer MDA-MB-231 cells expressing aluciferase reporter enzyme (MDA-MB-231-Luc) were obtained from ATCC(American Type Culture Collection) and cultured in Dulbecco's modifiedEagle's media (Invitrogen) and supplemented with 10% fetal bovine serum(Invitrogen), 100 μg/mL streptomycin, and 100 unites/mL penicillin(Invitrogen). The cells were maintained in a humidified incubator at 37°C. and 5% CO₂. A paclitaxel-resistant subline of the MDA-MB-231 cellline (MDA-MB-231.DR) was induced by chronic exposure of MDA-MB-231 cellsto 5 nM paclitaxel (PTX, Sigma Aldrich) with increasing concentration ateach passage over 8 weeks to reach a final concentration of 20 nM.Initially, cells were maintained at 5 nM PTX with increasingconcentration in increments of 5 nM after every other passage over 8weeks to reach a final concentration of 20 nM. Once resistance topaclitaxel was confirmed, the MDA-MB-231.DR cells were maintained at 5nM PTX.

Synthesis of mPEG(HZ)-Mal and RGD-PEG(HZ)-Mal

NHS-PEG-SH (MW 3400) and mPEG-HZ (MW 5000) were obtained from Nanocs,cRGDfk was obtained from Peptides International.

To synthesize the non-targeted, pH-cleavable PEG spacer,mPEG5000-hydrazide (Laysan Bio) was reacted with N-4-acetylphenylmaleimide (APM). First, 87.8 mg (MW=5000, 1 equivalent, 17.56 μmol) ofthe hydrazide-derivatized mPEG5000 was dissolved in 10 mL DCM/MeOH(50/50) and 100 mg Na2SO4 was added. Next, 11.7 mg (MW=215.2, 3.1equivalent, 54.44 μmol) of APM was dissolved in 1 mL DCM/MeOH (50/50)and added drop-wise into the mPEG-hydrazide solution. After the additionof APM, acetic acid (1.77 μL of 34% v/v solution in DCM, 0.6 equivalent,10.54 μmol) was added. The reaction was stirred for 24 hours at roomtemperature under nitrogen. After 24 hours, the solution wasprecipitated into cold diethyl ether (3×) to obtain a purified product.The H1 NMR spectrum (Solvent: CDCl3) of mPEG5000(HZ)-maleimide was usedto confirm the correct structure using the following characteristicpeaks: 8.43 (s, 1H, —NH—), 8.06 (d, 2H, in phenyl), 7.54 (d, 2H, inphenyl), 6.91 (2, 2H, two olefinic protons of maleimide), 3.48-3.58 (m,438H, PEG).

A three-step reaction was used to synthesize the cRGD-targetedpH-cleavable PEG-hydrazone moiety. First, cRGDfk was first conjugated tothe heterobifunctional NHS-PEG3400-SH: 25 mg (MW=603.7, 2 equivalent,41.4 μmol) of c(RGD)fk was dissolved in 5 mL DMF. Cyclic (RGD)fk wasused at 2× molar excess to the NHS-PEG3400-SH. Next, 70.4 mg (MW=3400, 1equivalent, 20.7 μmol) of NHS-PEG3400-SH was dissolved in 1 mL of DMFand added drop-wise into the c(RGD)fk/DMF solution. After addition, 100μL of DIPEA was added to the solution. The solution was stirred gentlyat room temperature for 4 hours. The solution was precipitated into anexcess of diethyl ether (3×) to remove excess c(RGD)fk and obtain thepurified cRGD-PEG3400-SH product. To ensure free thiol availability, 100mg of dithiothreitol (DTT) was added to the solution and stirredovernight to reduce any disulfide bonds present in the synthesizedcRGD-PEG-SH. Free DTT was removed using a desalting spin column (1.8KMWCO). The product was lyophilized, resuspended in chloroform and storedat −80° C. Conjugation of cRGD to PEG was confirmed by an observed shiftof ˜600 in the maldi-tof spectrum. To create a hydrazide-activatedcRGD-PEG, 25.2 mg (MW≈4000, 1 equivalent, 6.3 mol) of cRGD-PEG-SH wasdissolved 5 mL chloroform and reacted with 4.25 mg (N-ε-maleimidocaproicacid) hydrazide (EMCH, MW=225.24, 3 equivalents, 18.9 μmol), 4.39 μL oftrimethylamine was added to the reaction (TEA, 5 equivalents, 31.5μmol). The reaction was carried out for 4 hours at room temperatureunder stirring. After, the reaction solution was purified using a spincolumn (1.8K MWCO). The product was lyophilized, resuspended inchloroform and stored at −80° C. For the final step, 12 mg cRGD-PEG(HZ)(1 equivalent, 2.84 mol) was reacted with 1.2 mg N-4-acetylphenylmaleimide (APM, 2 equivalent, 5.68 μmol) overnight at room temperatureunder constant stirring. The reaction mixture was purified on a silicagel column using chloroform:methanol mobile phase (9:1 v/v). The finalproduct was concentrated and lyophilized. The H1 NMR spectrum (Solvent:DMSO) of cRGD-PEG3400(HZ)-maleimide was used to confirm the structurewith the following characteristic peaks: 8.48 (s, 1H, —NH—), 8.0 (d, 2H,in phenyl), 7.6-7.8 (m, cRGD), 7.45 (d, 2H, in phenyl), 7.2 (2, 2H, twoolefinic protons of maleimide), 3.1-3.5 (m, 304H, PEG).

Preparation of PEG-Modified ECO/siRNA Nanoparticles

The ECO lipid carrier was synthesized as described previously. ECO(MW=1023) was dissolved in 100% ethanol at a stock concentration of 2.5mM for in vitro experiments and 50 mM for in vivo experiments. The siRNAwas reconstituted in RNase-free water to a concentration of 18.8 μM forin vitro experiments and 25 μM for in vivo experiments. For in vitroexperiments, an siRNA transfection concentration of 100 nM was used.ECO/siRNA nanoparticles were prepared at an N/P ratio of 8 by mixingpredetermined volumes of ECO and siRNA for a period of 30 minutes inRNase-free water (pH 5.5) at room temperature under gentle agitation toenable complexation between ECO and siRNA. The total volume of water wasdetermined such that the volume ratio of ethanol:water remained fixed at1:20. For RGD-PEG(HZ)-modified ECO/siRNA nanoparticles, RGD-PEG(HZ)-Malwas first reacted with ECO in RNase-free water at 2.5 mol % for 30minutes under gentle agitation and subsequently mixed with siRNA inRNase-free water for an additional 30 min. RGD-PEG(HZ)-mal was preparedat a stock solution concentration of 0.32 mM in RNase-free water. Again,the total volume of water was determined such that the volume ratio ofethanol:water remained fixed at 1:20.

Nanoparticle Characterization

The zeta potential of unmodified, mPEG- and mPEG(HZ)-modified ECO/siRNAnanoparticle formulations at different pHs in PBS was determined with aBrookhaven ZetaPALS Particle Size and Zeta Potential Analyzer(Brookhaven Instruments). For each formulation, the nanoparticles werediluted in PBS solutions at pH 7.4, 6.5, or 5.4. The zeta potentialmeasurement was taken at each indicated time point up to 4 hours. Datarepresents the mean of three independently conducted experiments.

pH-Dependent Membrane Disruption Hemolysis Measurement

The hemolytic activity was measured to determine the membrane-disruptiveability of unmodified, mPEG- and mPEG(HZ)-modified ECO/siRNAnanoparticle formulations at pH levels corresponding to various stagesof intracellular trafficking. Red blood cells (RBCs) isolated from rats(Innovative Research Inc.) were diluted 1:50 in PBS solutions at pH 7.4,6.5, and 5.4. ECO/siRNA nanoparticles were prepared at a volume of 150μL and incubated with an equal volume of the various RBC solutions in a96-well plate at 37° C. for 2 hours. Following incubation, samples werecentrifuged and the absorbance of the supernatants was determined at 540nm. Hemolytic activity was calculated relative to the hemolytic activityof 1% Triton X-100 (Sigma Aldrich), a non-ionic surfactant. Each pH wasconducted in triplicate and the data presented represents the mean andstandard deviation.

Flow Cytometry for Nanoparticle Cellular Uptake Measurements

Cellular uptake and intracellular delivery of various ECO/siRNAnanoparticle formulations include mPEG-maleimide, mPEG(HZ)-maleimide,RGD-PEG-maleimide, RGD-PEG(HZ)-maleimide were evaluated quantitativelywith flow cytometry. The ECO/siRNA nanoparticle formulations wereprepared with 25 nM AlexaFluor647-labeled siRNA (Qiagen). Approximately2.5×10⁴ MDA-MB-231 cells were seeded onto 12-well plates and grown foran additional 24 hours. The cells were transfected with each ECO/siRNAnanoparticle formulation in 10% serum media. After 4 hours, thetransfection media was removed and each well was washed twice with PBS.The cells were harvested by treatment with 0.25% trypsin containing 0.26mM EDTA, (Invitrogen) collected by centrifugation at 1000 rpm for 5 min,resuspended in 500 μL of PBS containing 5% paraformaldehyde, and finallypassed through a 35 am cell strainer (BD Biosciences). Cellularinternalization of ECO/siRNA nanoparticles was quantified by thefluorescence intensity measurement of AlexaFluor 647 fluorescence for atotal of 10,000 cells per each sample using a BD FACSCalibur flowcytometer. Each formulation conducted in triplicate and the datapresented represents the mean fluorescence intensity and standarddeviation.

For studying the endocytic trafficking pathway, the following inhibitorswere used 1 hour prior to transfection in MDA-MB-231 cells with thevarious ECO/siRNA nanoparticles: 4° C., Cytochalasin D (5 μm/mL; SigmaAldrich), Genistein (200 μM; Sigma Aldrich), and Nocadozole (20 μM;Sigma Aldrich). After 1 hour, the various ECO/siRNA nanoparticlesformulated with the fluorescent AF647-labeled siRNA, as described above,were added to the cells. After an additional 2 hours, the cells wereharvested and processed as described above. Similarly, cellularinternalization of ECO/siRNA nanoparticles was quantified by thefluorescence intensity measurement of AlexaFluor 647 fluorescence for atotal of 10,000 cells per each sample using a BD FACSCalibur flowcytometer. Each formulation was conducted in triplicate and the datapresented represents the mean fluorescence intensity and standarddeviation.

Confocal Microscopy of Nanoparticle Uptake and Intracellular Release ofsiRNA

Live cell confocal microscopy was used to assess the cellular uptake,endolysosomal escape, and intracellular release of siRNA. Approximately1×10⁵ MDA-MB-231 cells were seeded onto glass-bottom micro-well dishes.After 24 hours, the cells were stained for 30 minutes each with 5 ag/mLHoechst 33342 (Invitrogen) and with 50 nM Lysotracker Green DND-26(Molecular Probes). RGD-PEG- and RGD-PEG(HZ)-modified ECO/siRNAnanoparticles were formed at an N/P ratio of 8 and a 25 nM siRNAconcentration with an AlexaFluor 647-labelled siRNA. Images were takenusing an Olympus FV1000 confocal microscope while the cells were housedin a humidified weather station under 5% CO₂.

In Vitro Luciferase Silencing Efficiency

MDA-MB-231-Luc cells were seeded in 24-well plates at a density of 2×10⁴cells and allowed to grow for 24 hours. Transfections were carried outin 10% serum media with an N/P ratio of 8 and 100 nM anti-luciferasesiRNA concentration (Dharmacon: sense sequence:5′-CUUACGCUGAGUACUUCGAdTdT-3′ (SEQ ID NO: 5), anti-sense sequence:5′-UCGAAGUACUCAGCGUAAGdTdT-3′ (SEQ ID NO: 6)). Following a 4 hourtransfection period, the media was replaced with fresh serum-containingmedia and the cells continued to grow for up to 72 hours. Forexperiments using chloroquine (Sigma Aldrich), transfections wereconducted in a similar manner either with or without 100 μM chloroquine.As above, following a 4 hour transfection period, the media was replacedwith fresh serum-containing media and the cells continued to grow for upto 48 hours. At each time point for luciferase silencing experiments,the cells were rinsed twice with PBS and lysed using the reporter lysisbuffered provided in the Promega Luciferase Assay kit. Following lysis,the cells were centrifuged at 10,000 g for 5 minutes and 20 μL celllysate was transferred to a 96-well plate. To quantify luciferaseexpression, 100 μL Luciferase Assay Reagent was added to each well andthe luminescence was read using a SpectraMax microplate reader(Molecular Devices). Luciferase activity was normalized to the totalprotein content measured from the cell lysate of each well using the BCAassay (Thermo Scientific). Data was presented relative to the control,which received no siRNA treatment.

In Vivo Luciferase Silencing Efficiency

MDA-MB-231-Luc cells were engrafted into the mammary fat pad of femalenude mice (2×10⁶ cells/mouse). Once the tumors reached an average of 250mm³, the mice were randomly sorted into 5 groups (n=3): 1) PBS control,2) PEG-ECO/siLuc, 3) PEG(HZ)-ECO/siLuc, 4) RGD-PEG-ECO/siLuc, 5)RGD-PEG(HZ)-ECO/siLuc. All siRNA nanoparticle variations were formulatedat 1.0 mg/kg siRNA in a total injection volume of 150 μL. All micereceived a single intravenous tail vein injection of the variousnanoparticle formulations following bioluminescent imaging on day 0.Expression of luciferase was quantified using bioluminescence imaging onday 0, 1, 3, 5, and 7. The bioluminescence signal intensity wasquantified from a region of interest (ROI) placed over the tumor area.Data was normalized to the average signal intensity of day 0.

Fluorescence Molecular Tomography

Fluorescence imaging of siRNA accumulation within primary MDA-MB-231mammary fat pad tumors was performed using the FMT 2500 quantitativefluorescence tomography system (Perkin-Elmer). Mice were treated with anintravenous tail vein administration of AlexaFluor 647-conjugated siRNA(1.0 mg/kg) with the various ECO nanoparticle formulations in a totalinjection volume of 150 μL. The mice were imaged before and afterintravenous injection of the nanoparticles at 30 min, 1 h, 2 h, 4 h, 8h, and 24 h.

Ex Vivo Flow Cytometry and Confocal Microscopy

Mice treated with AF 647-loaded ECO nanoparticles were sacrificed at 48hours post-injection whereupon the primary tumor was resected anddisaggregated into single cell suspensions using mechanical force anddisaggregation solution as described previously. The cell suspension wasstained with FITC-conjugated mouse MAb against human epithelial antigen(EpCAM) (HEA125; Miltenyi Biotec, Auburn, Calif.) in the dark and on icefor 10 minutes. After staining, the cells were washed and centrifuged,fixed with paraformaldehyde, and finally passed through a 35 m cellstrainer (BD Biosciences). Flow cytometry was conducted using thefluorescein channel for HEA-FITC and Cy5 channel for AF647-conjugatedsiRNA delivered by the ECO nanoparticles for a total of 10,000 cells pereach sample using a BD FACSCalibur flow cytometer. Gating within thefluorescein channel was used to identify EpCAM (+) and EpCAM (−)populations. Each formulation was conducted in triplicate and the datapresented represents the mean fluorescence intensity and standarddeviation. Following flow cytometry, the cell suspensions were examinedunder an Olympus FV100 confocal microscope.

Semi-Quantitative Real-Time PCR Analyses

Real-time PCR studies were performed as described previously. Briefly,MDA-MB-231 or MDA-MB-231.DR cells (100,000 cells/well) were seededovernight onto 6-well plates. The cells were then treated with ECOnanoparticles with a non-specific siRNA or eiF4E-specific siRNA (eIF4E:AAGCAAACCUGCGGCUGAUCU (SEQ ID NO: 7)). At each indicated time point,total RNA was isolated using the RNeasy Plus Kit (Qiagen) and reversetranscribed using the iScript cDNA Synthesis System (Bio-Rad).Semi-quantitative real-time PCR was conducted using iQ-SYBR Green(Bio-Rad) according to manufacturer's recommendations. In all cases,differences in RNA expression for each individual gene were normalizedto their corresponding GAPDH RNA signals.

Primer Sequences:

eIF4E: Sense (SEQ ID NO: 8) 5′-CTACTAAGAGCGGCTCCACCAC-3′; Antisense(SEQ ID NO: 9) 5′-TCGATTGCTTGACGCAGTCTCC-3′; GAPDH Sense (SEQ ID NO: 10)5′-ACGGATTTGGTCGTATTGGGCG-3′; Antisense (SEQ ID NO: 11)5′-CTCCTGGAAGATGGTGATGG-3′.Western Blot Analyses

Immunoblotting analyses were performed as previously described. Briefly,MDA-MB-231 and MDA-MB-231.DR cells were seeded into 6-well plates(1.5×10⁵ cells/well) and allowed to adhere overnight. The cells werethen treated with RGD-PEG(HZ)-ECO/siRNA complexes (N/P=8, siRNAconcentration of 100 nM) in complete growth medium. After 5 days,detergent-solubilized whole cell extracts (WCE) were prepared by lysingthe cells in Buffer H (50 mM β-glycerophosphate, 1.5 mM EGTA, 1 mM DTT,0.2 mM sodium orthovanadate, 1 mM benzamidine, 10 mg/mL leupeptin, and10 mg/mL aprotinin, pH 7.3). The clarified WCE (20 mg/lane) wereseparated through 10% SDS-PAGE, transferred electrophoretically tonitrocellulose membranes, and immunoblotted with the primary antibodies,anti-eIF4E (1:1000; Abcam) and anti-β-actin (1:1000; Santa CruzBiotechnology).

Cytotoxicity Assay

Cytotoxicity assays were performed in a 96-well plate as describedpreviously, by seeding 2,000 MDA-MB-231 or MDA-MB-231.DR cells/well.First, RGD-PEG(HZ)-modified ECO/siRNA nanoparticles were used totransfect MDA-MB-231 or MDA-MB-231.DR with either siNS or sieIF4E for 48hours. Next, the wells were washed twice with PBS and incubation withvarious concentrations of PTX in fresh media. After 2 additional days,the MTT reagent (Invitrogen) was added to the cells for 4 hours followedby the addition of SDS-HCl and further incubation for 4 hours. Theabsorbance of each well was measured at 570 nm using a SpectraMaxspectrophotometer (Molecular Devices). Cellular viability was calculatedas the average of the set of triplicates for each PTX concentration andwas normalized relative to the no treatment control. Drug resistance wasconfirmed with an MTT assay to determine the IC₅₀ of paclitaxel. TheIC₅₀ was defined as the dose of drug required to inhibit cell viabilityby 50%.

In Vivo Tumor Growth Inhibition Study

For in vivo anti-tumor efficacy studies, MDA-MB-231.DR cells (2×10⁶cells/mouse) were inoculated in the mammary fat pads of female nu/numice. When the tumors reached and average of 150 mm³, the mice wererandomly sorted into 4 groups (n=5): 1) PBS control, 2)RGD-PEG(HZ)-ECO/siNS (1.5 mg/kg siRNA)+PTX (5 mg/kg), 3)RGD-PEG(HZ)-ECO/sieIF4E (1.5 mg/kg siRNA)+PTX (5 mg/kg), 4)RGD-PEG(HZ)-ECO/sieIF4E (1.5 mg/kg siRNA). RGD-PEG(HZ)-ECO/siRNAnanoparticles were administered by intravenous injection into thelateral tail vein while PTX was administered in 10% DMSO/PBS with anintraperitoneal injection. Tumor growth was monitored by BLI and tumorsize was monitored with caliper measurements. Three days after the finaltreatment, the mice were sacrificed to harvest tumor tissues.

Bioluminescent Imaging

Longitudinal imaging of the mice was performed using the Xenogen IVIS100 imaging system. D-luciferin (Xenogen) was dissolved in PBS (15mg/mL), and 200 μL of the luciferin stock solution (15 mg/mL) wasinjected i.p. 5 minutes before measuring the light emission. Mice wereanesthetized and maintained under 2.5% isoflurane. Bioluminescentsignals were quantified using Living Image software (Xenogen) by drawingan ROI over the tumor area.

Immunofluorescence and Immunohistochemical Staining

For immunohistochemistry, primary tumor samples were embedded in optimumcutting temperature (O.C.T.) compound (Tissue-TeK; Torrence, Calif.) inpreparation for cryostat sectioning and immediately frozen. The sampleswere then sectioned, fixed in paraffin, and maintained at −80° C. Thesamples were stained with H&E to evaluate the presence of tumor tissue.Briefly, the samples were fixed in 10% formalin, rehydrated in 70%ethanol and rinsed in deionized water prior to hematoxylin staining.Samples were then rinsed in tap water, decolorized in acid alcohol,immersed in lithium carbonate and rinsed again in tap water. Next, theeosin counterstain was applied and slides were dehydrated in 100%ethanol, rinsed in Xylene and finally mounted on a coverslip withBiomount.

For immunofluorescence detection of eIF4E (Abcam: ab1126), survivin(Abcam: ab76424), Cyclin D1 (Abcam: ab16663), and VEGF (Abcam: ab46154),the paraffin-embedded slides were first deparaffinized using a series ofwashes in xylene and decreasing concentrations of ethanol. Heat-inducedantigen retrieval was performed using a pressure cooker in sodiumcitrate buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0) for 20minutes. Following heat-induced antigen retrieval, the samples wereblocked in TBST solution containing donkey serum and washed three timeswith TBST. The primary antibody was applied at dilution of 1:100 inblocking solution for 1 h followed by three rinses with TBST. The AlexaFluor 647 secondary antibody (Abcam: ab150079) was applied at a dilutionof 1:1000 in blocking solution for 1 h followed by three washes withTBST and counterstained with DAPI at a dilution of 1:2500 in blockingsolution. After washing with TBST and mounting in an anti-fade mountingsolution (Molecular Probes), the samples were imaged using a confocalmicroscope.

Toxicity, Immune Response, and Pathology Studies

Female BALB/c mice (Jackson Laboratories) were used to study thetoxicity and immune response of systemic treatment with ECO/siRNA andRGD-PEG(HZ)-ECO/siRNA nanoparticles. Following 1, 3 and 5 injections(n=5 for each amount of injections) spaced 5 days apart, blood wascollected at 2 h and 24 h post-injection. Plasma was isolated from bloodsamples using Microtainer tubes (Becton Dickinson). To measure plasmacytokine levels, TNFα, IL-6, IL1-2, INFγ were quantified by ELISAaccording to the manufacturer's instructions (Invitrogen).

Statistical Analyses

Statistical values were defined using unpaired Student's t-test, withp<0.05 considered to be statistically significant.

Results

Surface Modification of ECO/siRNA Nanoparticles with pH-Cleavable PEGLayer Restores Intrinsic pH-Sensitive Activity

The chemical structure of ECO contains various amino groups such that itcarriers a net positive charge at neutral pH. These amine groupselectrostatically complex with the siRNA cargo and contribute towardsthe pH-sensitive amphiphilic characteristic of the delivery system. Theamino groups, across a range of pKa's, become protonated in an acidicenvironment to promote pH-dependent membrane disruption to allow forendolysosomal escape. At an N/P ratio of 8, ECO/siRNA nanoparticlesexhibit a zeta potential of 22.3±1.73 mV at neutral pH. With increasingacidity, unmodified ECO/siRNA nanoparticles exhibit a pH-sensitive andtime-dependent increase in zeta potential correlating to the protonationof the cationic ethylenediamine head group (FIG. 8A). This protonationis hypothesized to enhance the electrostatic interactions between theECO carrier and the anionic membrane lipids to promote bilayerdestabilization to enable escape into the cytosol. It was observed thatmodification of the siRNA nanoparticle surface with mPEG3400 at a 2.5mol % through thiol-maleimide chemistry decreased the overall zetapotential to 12.3±1.39 mV (FIG. 8B). While unmodified ECO/siRNAnanoparticles exhibited pH-sensitivity at pH 6.5 and 5.4, this behaviorwas attenuated in PEG-modified nanoparticles. After 4 hours ofincubation in PBS solutions at pH 6.5 and 5.4, PEGylated ECO/siRNAnanoparticles carried a zeta potential of 17.4±1.1 mV and 18.6±2.9 mV,respectively, compared to 32.5±2.7 mV and 39.8±3.1 mV for unmodifiedECO/siRNA nanoparticles. This suggests that the surface aqueous phaseformed by PEGylation may impede the protonation of the cationic headgroup. This was confirmed with the observation that the pH-sensitivityis further diminished towards neutrality by increasing the PEG surfacedensity to 10 mol % (FIG. 9).

The insertion of the acid-labile hydrazone linkage within the PEG moietycreated a pH-cleavable PEG layer to restore the pH-sensitivity of thecore ECO/siRNA nanoparticles (FIG. 8C). Hydrazone is awell-characterized linkage known to hydrolyze at pH levels correspondingto the environment of the endolysosomal compartments. The hydrolysis ofhydrazone occurs when the —C═N nitrogen is protonated causing anucleophilic attack of water and the ultimate cleavage of the C—N bond.Previously, the hydrazone linkage has found popularity as a means toconjugate chemotherapeutics, such as doxorubicin, to a wide array ofdrug delivery systems and prodrugs to enhance intracellular release.This particular hydrazone-modified PEG linkage has also been usedpreviously to create PEG(HZ)-phosphatidylethanolamine conjugates capableof forming micelles. The hydrazone-based micelles were found to bestable at physiological pH but highly sensitive to mildly acidic pHs,resulting in robust degradation of micelles at pH 5.5. Similarly, theobserved increase in zeta potential at pH 6.5 and 5.4 presumablycorresponds to the acid-catalyzed hydrolysis of the hydrazone linkageand subsequent shedding of the PEG layer whereupon the cationicECO/siRNA core nanoparticle surface is exposed. Once exposed, theethylenediamine head group within the ECO/siRNA nanoparticles becomesprotonated. At pH 7.4, the zeta potential remains constant at −12 mVindicating that the PEG layer remains intact as a result of thehydrazone linkage stability. Importantly, the stability of surfacecharge at pH 7.4 suggests the ECO/siRNA nanoparticles will remainPEGylated within the bloodstream at the normal physiological pH.Following exposure to pH 5.4, the hydrazone bond is degraded to removethe PEG layer. In turn, the surface zeta potential of PEG(HZ)-ECO/siRNAnanoparticles gradually increases and after 4 hours is similar to thatof the unmodified ECO/siRNA nanoparticles, 36.3±1.9 mV and 39.8±2.8 mV,respectively. However, the time to reach the maximum zeta potential isprolonged, possibly due to the slower kinetics involved with thehydrolysis of the hydrazone linkage.

We have previously shown that the protonation of the cationic head groupof ECO is directly coupled with the ability to induce pH-sensitivemembrane disruption, a key step for successful and efficient cytosolicdelivery of siRNA. To compare the effect of pH-cleavable tonon-cleavable PEGylation, we examined the pH-sensitive hemolyticactivity of ECO/siRNA nanoparticles modified with both PEGylationstrategies (FIG. 9). Non-cleavable PEG-ECO/siRNA nanoparticles had asignificantly lowered ability to induce hemolysis at pH 6.5 and 5.4compared to unmodified ECO/siRNA nanoparticles. Again, this isindicative of the PEG layer inhibiting the interactions with the lipidmembrane of the blood cells and also the protonation of the cationichead group of ECO. In alignment, increasing the PEG surface densityfurther inhibited the hemolytic activity of the non-cleavable PEGylatednanoparticles. Conversely, the pH-cleavable PEG(HZ)-ECO/siRNAnanoparticles induced pH-sensitive hemolysis on par with unmodifiedECO/siRNA nanoparticles. As the hemolytic activity was evaluated 2 hoursfollowing exposure of the red blood cells to the nanoparticles, bothformulations of nanoparticles would have reached similar levels ofprotonation, as observed in FIGS. 8A and C, indicating the cleavage ofthe hydrazone linkage and subsequent shedding of the PEG layer.

pH-Cleavable RGD-PEG Modification Induces Potent In Vitro SilencingEfficiency

To facilitate ligand-specific uptake of the ECO/siRNA nanoparticles intothe target population of cells, a cyclic-RGD (RGD) peptide wasconjugated to both pH-cleavable and non-cleavable PEG moieties. Cellularuptake into MDA-MB-231 human breast cancer cells was quantified usingflow cytometry with nanoparticles formulated with fluorescently labelledsiRNA (FIG. 10A). The inclusion of the hydrazone linkage in bothRGD-targeted and non-targeted ECO/siRNA nanoparticles had no significantdifference in the ability of the nanoparticles to gain internalizationinto the cells when compared to the non-cleavable counterparts.Unmodified ECO/siRNA nanoparticles exhibited robust cellular uptake dueto strong ionic interactions with the anionic cellular membrane.PEGylation endows neutrality to the nanoparticle surface and impedessuch interactions with the cell surface. The addition of the RGDtargeting peptide enhanced the cellular uptake and internalization dueto specific binding of the RGD peptide to the α_(v)β3 integrin known tobe present on the cellular surface of the MDA-MB-231 cells.

Surface modification of nanoparticles has been shown to influence theendocytic mechanism through which they are internalized by cells.Further, the endocytic mechanism can dictate the fate of nanoparticleswithin the intracellular trafficking pathways. By using a series ofknown pharmacological inhibitors of various endocytic pathways, theprimary mechanism of uptake was elucidated for the various formulationsof ECO/siRNA nanoparticles in MDA-MB-231 cells: 1) incubation of cellsat 4° C. to inhibit energy-dependent endocytic mechanisms, 2)cytochalasin D is generally classified as an inhibitor ofmacropinicytosis/phagocytosis but recently has been implicated withinhibition of clathrin- and caveolae-mediated pathways, 3) Genisteininhibits caveolae-mediated endocytosis and 4) Nocodazole inhibitsclathrin-mediated pathways. PEGylated ECO/siRNA nanoparticles were foundto be internalized primarily via clathrin-mediate endocytosis whileRGD-targeted nanoparticles entered primarily via caveolae-mediatedendocytosis. Interestingly, unmodified ECO/siRNA nanoparticles werefound to rely on both energy-dependent and independent mechanismssuggesting that the ECO lipid may be able to directly fuse with thephospholipid membrane of cells. Nanoparticles internalized by Clathrin-and caveolae-dependent mechanisms are often transferred to the lysosomesfor degradation, therefore, the ability of both RGD-targeted andnon-targeted nanoparticles to escape from the endolysosomal pathway isan important step for achieving gene silencing. No significantdifference was observed in endocytic pathways between pH-cleavable andnon-cleavable surface modifications.

The in vitro luciferase silencing efficacy of the surface modifiedECO/siRNA nanoparticles was evaluated in the MDA-MB-231-Luc cell line(FIG. 10B). We have shown that unmodified ECO/siRNA nanoparticles inducepotent and sustained gene silencing upwards of 95% due to their abilityto readily become internalized, escape from the endolysosomal pathway,and release the cargo siRNA into the cytosol. Accordingly, ECO/siRNAnanoparticles achieved 92.33±2.08% luciferase silencing 72 hourspost-treatment in MDA-MB-231-Luc cells. The luciferase silencingefficiency was significantly inhibited upon non-cleavable PEGylation ofthe nanoparticles (PEG-ECO/siRNA) to only 23.45±1.52% after 72 hours.While decreased cellular uptake contributes to the attenuated silencingefficiency, our hemolysis assay data suggests PEGylation also interfereswith the ability of the nanoparticles to escape from the endolysosomalpathway (FIG. 9). Indeed, the silencing efficiency significantlyincreased to 47.86±10.59% when the pH-cleavable PEG was used whencompared to PEG-ECO/siRNA. This observation was highlighted with theaddition of the RGD targeting peptide. Non-cleavable RGD-PEG-ECO/siRNAnanoparticles only achieved 54.17±10.01% luciferase silencing while thepH-cleavable RGD-PEG(HZ)-ECO/siRNA formulation reached 83.64±4.58%luciferase silencing 72 hours post-treatment.

Inclusion of Hydrazone Linkage Enhances Endosomal Escape

The ability of the hydrazone linkage to enable successful endolysosomalescape was confirmed through live cell confocal imaging (FIG. 10C).MDA-MB-231 cells were transfected with pH-cleavable and non-cleavableRGD-targeted ECO nanoparticles formulated with a fluorescently labelledsiRNA while co-stained with Lysotracker (green) to visualize the acidiccompartments of the endosomes and lysosome. Images taken after 10minutes reveal that both RGD-targeted nanoparticle formulations havesimilar interactions with the cellular membranes. At 3 hours, bothformulations exhibit strong co-localization (yellow) with the lateendosomes and lysosomes, consistent with the intracellular traffickingof caveolae-mediated endocytosis. However, 6 hours post-treatment, thenon-cleavable RGD-targeted nanoparticles appear contained within theendolysosomes as evident by the co-localization of the siRNA andLysotracker fluorescent signal. In contrast, the pH-cleavableRGD-targeted nanoparticles appear to have successfully escaped from theendolysosomal pathway. Minimal co-localization of the siRNA andLysotracker signal is observed and the dispersed siRNA signal within thecytosol indicates the siRNA has been released from the nanoparticles, asseen with unmodified ECO/siRNA nanoparticles. The inability of thenon-cleavable RGD-targeted nanoparticles to escape into the cytosolfurther validates the observed discrepancy in gene silencing efficiencywhen compared with the pH-cleavable counterpart (FIG. 10C).

The endosomolytic agent chloroquine that causes endosome rupture andcontent release into the cytosol was used to further verify the role thehydrazone linkage plays in promoting escape. For both targeted andnon-targeted nanoparticles, the silencing efficiency of pH-cleavablenanoparticles was not affected by chloroquine whereas non-cleavablenanoparticles exhibited a significantly enhanced silencing efficiency.The data suggests that upon treatment with chloroquine, complexesmodified with the non-cleavable PEG that were trapped withinendolysosomes were then freed into the cytosol while nanoparticlesmodified with the pH-cleavable PEG moiety had already escaped from theendolysosomes.

Targeted pH-Sensitive Nanoparticles Exhibit Potent and Sustained In VivoGene Silencing

To study differences in the in vivo gene silencing efficiency of thedeveloped pH-cleavable surface modification strategy, the delivery ofanti-luciferase siRNA (1.0 mg/kg) following a single systemicadministration was investigated using bioluminescent imaging in aprimary mammary fat pad MDA-MB-231 breast cancer tumor model (FIGS. 10Aand B). Unlike the in vitro luciferase silencing findings, thenon-targeted ECO/siRNA nanoparticles, both pH-cleavable andnon-cleavable formulations, had a negligible silencing effect onluciferase expression, suggesting minimal cellular uptake of the siRNAinto the tumor cells. In contrast, both RGD-targeted formulationsinduced luciferase silencing to varying degrees for up to 7 days: thenon-cleavable RGD-targeted ECO achieved 52.04% luciferase silencingwhile the pH-cleavable RGD-targeted formulation achieved 85.17%silencing compared to the no treatment control on day 7. This trend isconsistent with the enhancement of silencing efficiency observed invitro (FIG. 10C).

Targeting Ligand Enhances Tumor Retention of Nanoparticles by PromotingInternalization within Tumor Cells

The tumor localization and subsequent delivery of functional siRNAfollowing systemic administration of the various siRNA nanoparticleformulations was investigated. Fluorescence molecular tomography (FMT)enabled the analysis of tumor accumulation and retention of thenanoparticle-delivered siRNA over time. Mice received a singlesystemically administered dose of fluorescently-labeled siRNA (AF 647)delivered by the various surface modified ECO/siRNA nanoparticleformulations. Recent studies have demonstrated that the presence of atargeting ligand does not necessarily impact the initial tumoraccumulation of the nanomedicine delivery system, but rather mayfacilitate tumor cell targeting resulting in longer tumor retention. Inalignment, longitudinal FMT imaging studies revealed that the initialtumor localization of siRNA delivered by the targeted and non-targetednanoparticles was not significantly different for the first 4 hoursfollowing systemic administration (FIGS. 11C and D). By 8 hours however,RGD-targeted nanoparticles were observed to accumulate within the tumorto a greater extent than the non-targeted formulations, regardless ofwhether the PEG modification was pH-cleavable or not. At 24 hours, bothRGD-targeted nanoparticle formulations were retained within the tumorwhile the non-targeted formulations appeared to be washed out, due inpart to the inability of the non-targeted nanoparticles to promotecellular internalization, as evident by the minimal presence of siRNAsignal from the tumor ROI.

Active targeting of the ECO/siRNA nanoparticles using the RGD peptidemay aid in the specific selection of cancer cells within the tumor siteand promote internalization of the nanoparticles to a greater extentinside the target cells compared to non-target cells. To evaluate ifselective uptake dependent upon cell type occurred, ex vivo flowcytometry was used to quantify siRNA internalization to studydifferences in cellular uptake of the targeted and non-targetednanoparticles between human tumor and murine stromal cells. At 48 hours,tumors from the systemically treated mice were excised, disaggregatedinto single cell suspensions, and stained for the epithelial cellularadhesion molecular (EpCAM) using HEA-FITC to distinguish between thehuman MDA-MB-231 cancer cells and the murine stromal cells. FACSanalysis of the cell suspensions in the FITC channel revealed twodistinct cellular populations: EpCAM (+), human cancer cells and EpCAM(−), murine stromal cells. Gating for EpCAM (−) cells in the AlexaFluor647 channel revealed a minimal shift in fluorescence between bothtargeted and non-targeted nanoparticle formulations compared to the PBSnegative control. A distinct shift was observed in EpCAM (+) cells fortargeted nanoparticles compared to both the non-targeted and PBS controlgroups, suggesting that the RGD-targeted nanoparticles are internalizedmore efficiently and preferentially by the human cancer cells. Contourplots highlight the two distinct EpCAM (+) and EpCAM (−) cellularpopulations. While the siRNA signal from non-targeted nanoparticleformulations was evenly distributed throughout both populations, thesiRNA signal in EpCAM (+) cells was markedly higher for the RGD-targetednanoparticle formulations. No difference was observed between pH- andnon-cleavable formulations (data not shown).

Following flow cytometry, the cell suspensions were observed under aconfocal microscope. Microscopy analysis revealed both pH-cleavable andnon-cleavable RGD-targeted nanoparticle formulations were readilyinternalized by EpCAM (+) cells whereas the non-targeted formulationsshowed minimal uptake. While the targeted ECO/siRNA nanoparticles didnot initially transport the siRNA cargo to the tumor site moreefficiently than non-targeted nanoparticles according to the FMT data(FIG. 11D), it appears that RGD peptide enabled cell-specificrecognition and internalization of the siRNA. The enhanced cellularuptake correlated to the prolonged retention of fluorescent signalwithin the tumor ROI as determined by FMT (FIG. 11D) and also thesustained luciferase silencing efficiency of the targeted nanoparticles(FIGS. 11A and B). As internalization of siRNA from pH-cleavable andnon-cleavable RGD-targeted nanoparticle formulations appeared similaraccording to FMT and ex vivo confocal microscopy, the improvement insilencing efficiency of the pH-cleavable formulation may be a directresult of enhanced endolysosomal escape.

A surface modification strategy that combines PEGylation and activetargeting not only allows the ECO/siRNA nanoparticles to retain stealthproperties during circulation and accumulate at the tumor site bypassive targeting, but the PEG shielding moiety provides a tether forversatile incorporation of various active targeting ligands. Whenfurther combined with the pH-cleavable hydrazone linkage, the developedsurface modification strategy may significantly improve the therapeuticefficacy of the ECO/siRNA nanoparticle delivery system compared to thenon-targeted and non-cleavable systems. Taken together with the in vivoluciferase silencing data, the differences in silencing efficiencyacross all formulations suggest: i) targeted nanoparticles areinternalized to a greater extent by the tumor cells and ii) onceinternalized, formulations with the pH-cleavable PEG modification aremore effective in delivering siRNA into the cytosol due to enhancedendolysosomal escape.

Silencing eIF4E by RGD-Targeted pH-Cleavable PEG-Modified ECO/siRNANanoparticles Enhances Sensitivity to Paclitaxel in a Drug-ResistanceTriple-Negative Breast Cancer Cell Line

The ability of the pH-cleavable RGD-PEG(HZ)-ECO/siRNA nanoparticles tomediate knockdown of eIF4E mRNA and protein was next evaluated in vitro.To study the therapeutic consequence of eIF4E downregulation ondrug-resistant breast cancer cells, a paclitaxel-resistant subline ofthe MDA-MB-231 cell line (MDA-MB-231.DR) was induced by chronic exposureof MDA-MB-231 cells to paclitaxel. Interestingly, PTX-resistant cellswere found to upregulate eIF4E expression (FIGS. 12A and B). While awidely used chemotherapeutic agent, PTX has been shown to activatesignaling pathways that both promote and inhibit cell death. Normally,binding of 4E-BP1 to eIF4E will prevent eIF4E from binding to eIF4G andinitiating cap-dependent translation. PTX has been found to diminish thesuppressive role of 4E-BP1 through hyperphosphorylation to reduce theassociation affinity with eIF4E and promote its release. By doing so,eIF4E activity is elevated through a regained association with eIF4G andinitiation of translation. Along these lines, treatment of MDA-MB-231cells with PTX has been demonstrated elsewhere to also increase eIF4Eexpression in a dose-dependent manner. Importantly, treatment of bothcell lines with sieIF4E delivered by targeted and pH-cleavablenanoparticles was able to sustain potent eIF4E mRNA and proteinsilencing for upwards of 5 days. Nanoparticles delivering a non-specificsiRNA induced no significant downregulation of eIF4E expressionsuggesting that all silencing activity was due to the siRNA and not theECO carrier.

The therapeutic response of eIF4E silencing on enhancing the sensitivityof breast cancer cells to paclitaxel was determined by quantifying thecell viability after a combination of eIF4E silencing by the siRNAnanoparticles followed by treatment with PTX (FIG. 12C). Cells werefirst treated with RGD-PEG(HZ)-ECO/siRNA nanoparticles for 48 hoursfollowed by treatment with varying concentrations of PTX for anadditional 48 hours. For all conditions, cell viability decreased in aPTX concentration-dependent manner. The acquisition of resistance to PTXin the MDA-MB-231.DR subline was evident by a shift in the dose-responsecurve and the IC₅₀. A clear re-sensitization of MDA-MB-231.DR cells toPTX was observed when eIF4E was down-regulated using siRNA delivered byRGD-PEG(HZ)-ECO/siRNA nanoparticles. For example, at 0.5 ng/mL PTX,treatment with PTX alone induced 44.6±6.6% viability in MDA-MB-231 cellsand 80.7±2.9% viability in the drug-resistant subline. When coupled withsilencing of eIF4E, viability dropped to 7.5±5.4% and 26.3±3.6% inMDA-MB-231 and MDA-MB-231.DR, respectively, using the same concentrationof PTX.

Combination of siRNA Targeting eIF4E and Paclitaxel Inhibits PrimaryTumor Growth of Drug-Resistant MDA-MB-231 Cells

The ability of the pH-cleavable RGD-PEG(HZ)-modified ECO/siRNAnanoparticles to re-sensitize PTX-resistant MDA-MB-231.DR tumors wasevaluated in vivo. Female nude mice were engrafted with MDA-MB-231.DRcells in the mammary fat pad. When the tumors reached approximately 150mm³ in volume, the mice began receiving alternating treatments withsiRNA nanoparticles (1.5 mg/kg siRNA dose) and PTX (5 mg/kg) every 6days. Treatment with nanoparticles delivering non-specific siRNA (siNS)in combination with PTX had no inhibitory effect on tumor growth. Infact, after several weeks of treatment, the tumors of mice treated withsiNS+PTX grew larger and more rapidly compared to the no treatmentcontrol, as determined by bioluminescent imaging and tumor volumemeasurements (FIG. 13A-C). After 42 days of treatment, the no treatmentcontrol tumors were an average of 430.1±37.8 mm³ in volume and weighed404.9±43.6 mg whereas siNS+PTX tumors were 560.3±46.2 mm³ in volume andweighed 715.3±101.5 mg (FIG. 13C-E). Treatment of tumors withnanoparticles delivering anti-eIF4E siRNA alone significantly attenuatedtumor growth to 240.4±35.7 mm³ and 256.8±33.5 mg at day 42. This findingis supported by previously established data which demonstrates thatsilencing of eIF4E induces cell growth arrest. Likewise, Phase Iclinical evaluation of the LY2275796 antisense oligonucleotide targetingeIF4E found only cytostasis, and not anti-tumor activity, was achievedupon eIF4E downregulation. A combination of sieIF4E and PTXsignificantly inhibited tumor growth and lead to tumor regression,50.2±35.7 mm³ and 103.2±67.4 mg. Significant knockdown of eIF4E mRNA wasobserved in both groups of mice treated with RGD-PEG(HZ)-ECO/sieIF4Enanoparticles (FIG. 13F).

Interestingly, from Immunofluorescent staining of primary tumor samples,siNS+PTX-treated tumors exhibited elevated levels of eIF4E, survivin,cyclin D1 and VEGF mRNA compared to the no treatment group, suggestingthat treatment of drug-resistance cancers with PTX can further drivecancers to become more aggressive (FIG. 13C-E). This may occur throughactivation of the (PI3K)/akt signaling pathway implicated with both cellsurvival and drug resistance. As PTX relies upon apoptosis to inducetumor regression, elevated expression of survivin, an antiapoptoticfactor, may contribute to the observed resistance to PTX therapy:experimental upregulation of survivin has been shown to confer taxolresistance. A downregulation of survivin exhibited in treatment groupsreceiving sieIF4E may help suppress tumor growth by increasing thesusceptibility of cancer cells to apoptosis, particularly when coupledwith PTX therapy. Tumor growth may also be suppressed through areduction of angiogenesis, a consequence of VEGF downregulation alsoobserved in groups receiving sieIF4E. These data in conjunction with thesignificant tumor regression observed for the sieIF4E+PTX treatmentgroup indicates that knockdown of eIF4E re-sensitized the MDA-MB-231.DRcells to the cytotoxic effect of PTX.

Chemoresistance is a limitation in the treatment of patients with TNBC,where the lack of effective targeted therapies has left small-moleculebased strategies as the sole option. Molecular targets exploited fortargeted therapy against drug-resistant TNBCs could contributesignificantly to an improved standard of care. While eIF4E has beenexplored as a therapeutic target in TNBCs before, the presented studyrepresents the first evaluation of eIF4E in a drug-resistance TNBC cellline, therefore adding to the promising body of work surrounding eIF4E.

Long-Term Systemic Administration of RGD-PEG(HZ)-ECO/siRNA NanoparticlesElicits No Chronic Immune Response or Organ Damage

A concern of an RNAi-based approach to eIF4E therapy is the implicationof non-specific silencing of eIF4E in healthy tissues. While theinclusion of the RGD-targeting peptide can enhance selective uptakewithin tumor cells, accumulation of the siRNA nanoparticles innon-specific tissues may lead to eIF4E downregulation outside of thetumor. In the current study, the expression of eIF4E following treatmentwith the siRNA nanoparticles was not evaluated in other vital organs,however, it has been reported elsewhere that no systemic toxicity wasobserved when 80% knockdown of eIF4E was achieved in essential organsusing antisense technology. This may be explained by the phenomenonknown as oncogene addition whereby cancer cells are overly dependent onthe expression of a single gene for continued survival andproliferation. Under normal conditions, eIF4E is likely to be inactivedue to being bound to the inhibitory 4E-BPs. A reduction in eIF4E levelswould therefore have a minimal effect. Cancer cells characterized withelevated eIF4E expression, however, are more dependent on eIF4Eexpression than non-malignant cells or cancer cells with normal eIF4Eexpression.

Histopathological examination of liver and kidney tissues from micereceiving the long-term PTX and nanoparticle treatments was performed byhematoxylin and eosin (H&E) staining. No substantial toxicity or tissuedamage to the liver or kidney was observed across all treatment groups.The absence of toxicity from treatment groups who received multipleadministrations of PTX may be attributed to the relatively low andinfrequent dosing: 5 mg/kg every 6 days compared to the conventionaldose of 10 mg/kg. Treatment groups who received sieIF4E treatment alsorevealed no structural damage to the tissues, suggesting any systemicsilencing of eIF4E harbors minimal toxicity.

As nude mice bear an inhibited immune system, such animals are not asuitable model to study potential immunogenic responses followingsystemic administration of ECO/siRNA nanoparticles. Accordingly,immunocompetent BALB/c mice were used to study the possible immuneresponse following repeated tail-vein injections forRGD-PEG(HZ)-modified ECO/siRNA nanoparticles. Blood was collected at 2 hor 24 h following injections following 1, 3 and 5 injections spaced 5days apart. Systemic treatment with unmodified ECO/siRNA nanoparticleselicited a robust activation of all cytokines measured at both the 2 hand 24 h timepoints due to the high surface charge of the nanoparticles.PEGylation in the form of RGD-PEG(HZ)-modification significantlyattenuated the immune response for all tested cytokines. While cytokinelevels increased at 2 h for IL-6, IL-12 and IFN-γ, the serum levels werereduced to basal levels at 24 h, indicative of a transient response.Importantly, serum levels were not compounded over the course of 5repeated injections. These data, in conjunction with pathologicalexamination of the liver and kidneys are indicative of the long-termsafety of PEGylated ECO/siRNA nanoparticles.

Example 3

In this Example, we implemented the multifunctional amino lipid carrierECO,(1-aminoethyl)iminobis[N-oleicylcysteinyl-1-aminoethyl)propionamide],which can form stable self-assembly nanoparticles with therapeuticnucleic acids, including siRNA, DNA, and CRISPR/Cas. FIG. 14 illustratesthe schematic for the formation and working of the nanoparticle system.ECO undergoes electrostatic complexation with siRNA to form ECO/siRNAnanoparticles that are further stabilized by the hydrophobiccondensation of the oleic acid tails and auto-oxidation of cysteineresidues to form disulfide bonds. The ECO/siRNA nanoparticles can alsobe functionalized by conjugation with polyethylene glycol (PEG) forimproved biocompatibility and with cyclic RGD peptide for tumortargeting for in vivo gene delivery (FIG. 14a ). The nanoparticles areknown to mediate efficient cytosolic siRNA delivery and robust knockdownof target mRNAs through the PERC mechanism: pH-sensitive amphiphilicendosomal escape, followed by reductive dissociation and cytosolicrelease of the siRNA cargo, to enable effective RNAi (FIG. 14b ).

Here, we demonstrate efficient and effective nanoparticle-mediatedtargeting of the onco-lncRNA DANCR for TNBC therapy. We established thesignificant overexpression of DANCR in human breast cancer (BCa) cells,tumors, and The Cancer Genome Atlas (TCGA) database. We theninvestigated DANCR as a therapeutic target for effective TNBC therapy intwo cell lines and mouse models by silencing its expression usingtumor-targeting RGD-PEG-ECO/siDANCR nanoparticles to facilitateefficient cytosolic delivery of siDANCR. Transfections ofRGD-PEG-ECO/siDANCR nanoparticles showed significant and prolonged DANCRsilencing and inhibited invasion and proliferation of TNBC cells.Systemic injections of the RGD-PEG-ECO/siDANCR nanoparticles led tosuppression of TNBC proliferation in mice with high efficacy and noovert side-effects. We also investigated the mechanism of action ofDANCR and observed its pleiotropic roles in regulating multiplecancer-associated signaling pathways in TNBC.

Materials and Methods

Cell Lines and Culture

Normal human mammary epithelial cells (HMECs), hormone receptor positivebreast cancer cell lines MCF7 (ER⁺, PR^(+/−), Her2⁻) and ZR-75-1 (ER⁺,PR^(+/−), Her2⁺), and triple-negative breast cancer cell lines, namely,MDA-MB-231, Hs578T, and BT549 (claudin-low ER⁻, PR⁻, Her2⁻), werepurchased from ATCC (Manassas, Va.). HMECs were passaged in MammaryEpithelial Cell Basal Medium supplemented with components of the MammaryEpithelial Cell Growth Kit (Lonza, Allendale, N.J.). MCF7 and ZR-75-1cells were cultured in Eagle's Minimum Essential Medium (EMEM)supplemented with 10% FBS, 1% Penicillin/Streptomycin and 0.01 mg/mLrecombinant human insulin (Sigma, St. Louis, Mo.). MDA-MB-231, Hs578T,and BT549 cells were maintained in Dulbecco's Modified Eagle's Medium(DMEM) supplemented with 10% fetal bovine serum (FBS) and 1%Penicillin/Streptomycin. All the cells were grown at 37° C. and 5% CO₂.The cell lines were tested for the absence of mycoplasma using theMycoAlert™ Mycoplasma Detection Kit (Lonza).

TCGA Analysis

RNA-Seq data from TCGA database for DANCR transcript (ENST00000411630)was mined from the lncRNAtor website. Graphpad Prism was used to performdifferential gene expression analysis between 104 normal and 790 breastcancer samples (comprising 80 TNBC, 584 ER⁺, 508 PR⁺, 128 Her2⁺samples). Graphpad Prism was also used to construct dot plots and heatmaps, and perform statistical analyses for the expression data.

cDNA Array

Breast cancer cDNA Array IV (BCRT104) was purchased from OrigeneTechnologies (Rockville, Md.). DANCR expression was quantified byqRT-PCR using SyBr Green PCR Master Mix (Applied Biosystems, CA) andDANCR-specific primers, with 18S primers as controls. Gene expressionwas analyzed by the 2^(−ΔΔCt) method with 18S expression as the control.

Nanoparticle Formulation and Transfections

ECO-PEG-RGD/siRNA nanoparticles were formulated as previously described.Briefly, the amino lipid ECO (5 mM stock in ethanol) was gently agitatedwith RGD-PEG-Mal (RGD-PEG/ECO=2.5 mol %) in nuclease-free ultra-purewater for 30 min at RT. This was followed by complexation with siDANCRor siLuc (as negative control NC) at a final siRNA concentration of 100nM and N/P=10 for an additional 30 min to enable self-assembly formationof ECO-PEG-RGD/siDANCR or ECO-PEG-RGD/NC nanoparticles, respectively.For transfections, the nanoparticle formulation was mixed with culturemedia and added on to plated cells for 24-48 h, depending on therelevant experiments. The siDANCR duplex [sense 5′-GGU CAU GAG AAA CGUGGA UUA CAdCdC-3′ (SEQ ID NO: 12) and antisense 5′-GGU GUA AUC CAC GUUUCU CAU GAC CUC-3′ (SEQ ID NO: 13)] was purchased from IDT (Coralville,Iowa). The siLuc duplex [sense 5′-CCU ACG CCG AGU ACU UCG AdTdT-3′ (SEQID NO: 14) and antisense 5′-dTdT GGA UGC GGC UCA UGA AGC U-3′ (SEQ IDNO: 15)] was purchased from Dharmacon (Lafayette, Colo.).

Nanoparticle Characterization

The nanoparticles were diluted in nuclease-free water and characterizedfor particle size and zeta potential on the LiteSizer™ 500 (Anton Paar),according to manufacturer's instructions. The morphology of thenanoparticles was determined using Transmission electron microscopy(TEM), as described previously. In short, the nanoparticle suspension(20 μL) was pipetted onto a 300-mesh copper grid coated with a thinamorphous carbon film (20 nm). After blotting away excess sample with afilter paper, 3 μL of 2% uranyl acetate solution was added to stain thesamples. After two rounds of staining, the samples were dried and imagedvia TEM.

RNA Extraction and Analysis

Total RNA was extracted from cells and tissues using the RNeasy PlusMini Kit (Qiagen, Germantown, Md.), according to manufacturer'sinstructions. Reverse transcription was performed using the miScript IIRT Kit (Qiagen) and qPCR was performed using the SyBr Green PCR MasterMix. Gene expression was analyzed by the 2^(−ΔΔCt) method with 18Sexpression as the control. The following primer sequences wereused—DANCR: Fwd 5′-GCGCCACTATG TAGCGGGTT-3′ (SEQ ID NO: 16) and Rev5′-TCAATGGCTTGTGCCTGTAGTT-3′ (SEQ ID NO: 17); 18S: Fwd 5′-TCAAGAACGAAAGTCGGAGG-3′ (SEQ ID NO: 18) and Rev 5′-GGACATCTAAGGGCATCACA-3′ (SEQID NO: 19); EZH2: Fwd 5′-AGGA CGGCTCCTCTAACCAT-3′ (SEQ ID NO: 20) andRev 5′-CTTGGTGTTGCACTGTGCTT-3′ (SEQ ID NO: 21); Survivin: Fwd5′-ATGGCCGAGGCTGGCTTCATC-3′ (SEQ ID NO: 22) and Rev5′-ACGGCGCACTTTCTTCGCAGTT-3′ (SEQ ID NO: 23).

Viability Assay

MTT assay was performed according to manufacturer's protocol (ThermoFisher Scientific, Waltham, Mass.). Briefly, 5000 cells were plated on96-well plates. After 24 h, the cells were transfected with therapeuticand negative control nanoparticles. After 48 h, MTT agent (5 mg/mL) wasadded into the wells and incubated for 4 h. The purple crystals weredissolved in SDS-HCl by incubation for 4 h. The absorbance was measuredon SpectraMax microplate reader at 570 nm. Viability of control cellswas normalized to 100%, for comparison to treated cells.

BrdU Incorporation Assay

BrdU incorporation was measured using the BrdU Cell Proliferation ELISAKit from Abcam (Cambridge, Mass.), according to manufacturer'sinstructions. Briefly, 5000 cells were plated on a 96-well plate for 24h and then transfected with the nanoparticles. After 40 h, diluted BrdUreagent was added to the cells. After 8 h, the cells were fixed for 30min and incubated with anti-BrdU antibody for 1 h. After 3 washes, thecells were then incubated with Peroxidase Goat Anti-mouse IgG conjugatefor 30 min. Finally, TMB Peroxidase Substrate was added for colordevelopment (30 min), followed by the Stop Solution. Absorbance wasmeasured at 450 nm using SpectraMax Microplate reader. Viability ofcontrol cells was normalized to 100%, for comparison to treated cells.

Scratch Wound and Transwell Migration Assays

For the scratch wound assay, approximately 5×10⁵ TNBC cells were platedon 6-well plates for 24 h. A single straight scratch was made in themonolayer with a small pipet tip. After washing the detached cells withPBS, the cells were transfected with therapeutic and controlnanoparticles and monitored for up to 48 h, or until the scratch woundswere closed. Images were taken using the Moticam T2 camera.

For Transwell migration assays, 1-2×10⁵ cells transfected withnanoparticles were plated on Transwell inserts (VWR, Radnor, Pa.) coatedwith 0.28 mg/mL Corning™ Matrigel™ Membrane Matrix (Corning, N.Y.). Thenext day, the un-migrated cells in the inserts were removed by swabbingwith Q-tips. The migrated cells were fixed by treating the inserts with10% formalin for 10 min. After washing with PBS three times, the insertswere placed in 0.05% crystal violet stain for 20 min to stain themigrated cells. The inserts were washed under running water to removeexcess stain and set to dry overnight. Images of the purple migratedcells were taken using the Moticam T2 camera. The cells in the imageswere counted using the ImageJ software.

3D Tumor Spheroid Growth

For the Matrigel growth assay, 3-4×10⁵ TNBC cells were transfected withthe nanoparticles, suspended in 5% Matrigel™-containing media and platedon to 24-well plates coated with a thick layer of Corning™ Matrigel™Membrane Matrix. The ability of the cells to form tumor spheroids in the3D Matrix was monitored and photographed for up to 7 days using theMoticam T2 camera.

Western Blot

Total cellular protein was extracted. Protein extracts (40 ag) wereseparated by SDS-PAGE, transferred onto nitrocellulose membrane andimmunoblotted with primary antibodies overnight. The following primaryantibodies were purchased from Cell Signaling Technology (Danvers,Mass.)-: anti-β-catenin, anti-ZEB 1, anti-N-cadherin, anti-EZH2,anti-SUZ12, anti-me³-H3K7, and anti-survivin. After secondary antibodyincubation, the membranes were developed using ChemiDoc™ XRS+ Imager(Biorad, Hercules, Calif.). β-Actin was used as the loading control.

RNA Immunoprecipitation (RIP)

Magna RIP kit and DZNep were purchased from Millipore Sigma (Burlington,Mass.). RIP was performed according to manufacturer's instructions.Briefly, cell lysates were incubated with antibody-bead mix overnight at4° C. Following this, proteins were degraded and the RNA was isolated bythe phenol-chloroform extraction method. The RNA was precipitated usingKit components and ethanol at −80° C. overnight. For EZH2 inhibition,cells were treated with 500 μM DZNep for 72 h before harvesting for RIPassay.

Phosphokinase Array

The differential phosphorylation status of 43 different kinases and 2whole proteins with and without DANCR silencing was detected using theProteome Profiler Human Phospho-Kinase Array Kit (RnD Systems,Minneapolis, Minn.). MDA-MB-231 and BT549 cells were transfected withRGD-PEG-ECO/NC and RGD-PEG-ECO/siDANCR nanoparticles. After 48 h, thecells were lysed and the lysates were analyzed for the relativephosphorylation profiles, according to the manufacturer's instructions.The expression was quantified by subtracting the background signal fromthe mean pixel intensity of the duplicate spots and plotted as bargraphs.

Animal Models

Nude athymic mice (6-week-old nu/nu females) were purchased from theAthymic Animal and Preclinical Therapeutic Facility of the CaseComprehensive Cancer Center and housed in the Case Center for ImagingResearch at CWRU. All animal experiments were performed according to theprotocol and guidelines laid down by the IACUC and ARC of CWRU. Fortumor xenografts, 2×10⁶ MDA-MB-231 cells and 4×10⁶ BT549 cells suspendedin Matrigel-PBS (6 mg/mL) were injected into the mammary fat pads ofeach nude mouse. Tumor volumes were monitored and measured once a weekusing a Vernier caliper. When the average tumor volumes reached 70-90mm³, mice were randomized into control and treatment groups (n=6 forMDA-MB-231 and n=4 for BT549).

Tumor Xenograft Treatments

For in vivo therapy, nanoparticles were formulated as follows: ECO (50mM stock) was conjugated to RGD-PEG-Mal (RGD-PEG/ECO=2.5 mol %) for 30min, followed by complexation with siDANCR or siLuc (siRNA dose=1 mg/kg,[siRNA]=0.3 mg/mL) at N/P=8 for 30 more min, to obtainECO-PEG-RGD/siDANCR or ECO-PEG-RGD/NC nanoparticles, respectively. Theformulations were made in nuclease-free ultra-pure water and injectedinto the tail vein at siRNA dose of 1.0 mg/kg (100 μL per mouse) once aweek for 6 weeks. Tumor volumes and body weights of mice were monitoredonce a week for 6 weeks. At the end of the experiment, the animals wereeuthanized, and the tumors and vital organs were harvested for analysisof morphology, histology, and metastasis. Portions of the tissues werefixed in 10% neutral buffered formalin, followed by paraffin embedding,sectioning, and H&E staining. Staining and IHC services were provided bythe Tissue Resources Core Facility of CWRU. All the slides were reviewedby a certified pathologist.

Statistical Analyses

All the experiments were independently replicated at least 3 times(n=3), unless otherwise stated. Data are represented as mean±s.e.m.Statistical analysis was performed using Graphpad Prism. Data betweentwo groups was compared using unpaired Student's t-test. Data betweenthree groups was compared using One-way ANOVA. p<0.05 was considered tobe statistically significant.

Results

DANCR is Overexpressed in Triple-Negative Breast Cancer (TNBC)

We examined the endogenous levels of DANCR in a wide range of human BCacells, tumors, and healthy tissues. Using qRT-PCR, we compared theexpression of DANCR in BCa cell lines with distinct molecular profiles:hormone receptor (HR)-positive MCF7 and ZR-75, and TNBC Hs578T, BT549,and MDA-MB-231 cells. Compared to normal human mammary epithelial cells(HMECs), the BCa cells showed significantly elevated (4-9 fold) levelsof DANCR (FIG. 15a ). Among the cancer lines, the TNBC cells(particularly, BT549 and MDA-MB-231) overexpressed DANCR more (2-fold)than the HR-positive cells. Next, we quantified DANCR levels using acDNA array and found that DANCR is significantly upregulated (40-fold)in TNBC tumors than in normal breast tissues (FIG. 15b ). Analysis ofthe RNA-Seq data from TCGA database for DANCR expression in 104 normaland 790 BCa samples showed consistent results, where

DANCR was significantly overexpressed in BCa patients (FIG. 15c ), andparticularly in TNBC patients (FIG. 15d ), compared to healthy controls.The heat map in FIG. 15e shows a diverse range of DANCR expression inthe individual normal (N) and TNBC (T) patients, indicating theheterogeneity of the lncRNA. An additional comparison revealed higherDANCR expression in TNBC samples than in the ER⁺, PR⁺, and Her2⁺ tumors(1.34-fold, 1.33-fold, and 1.27-fold, respectively) (FIG. 15f-h ). Theseresults demonstrate that DANCR is substantially upregulated in BCa, andespecially more so in TNBC.

Targeted RGD-PEG-ECO/siDANCR Nanoparticles Facilitate Robust andProlonged DANCR Silencing in TNBC Cells

To effectively downregulate DANCR expression in TNBC, we formulatedstable targeted nanoparticles using the lipid ECO and unmodified siDANCRvia self-assembly. The nanoparticles were functionalized with cyclic RGDpeptide via a PEG spacer (3,400 Da) for tumor targeting and improvedbiocompatibility during systemic delivery. These RGD-PEG-ECO/siDANCRnanoparticles mediate efficient cytosolic siRNA delivery and robustknockdown of target mRNAs through the PERC mechanism (FIG. 14).RGD-PEG-ECO/siDANCR nanoparticles were formulated ([siRNA]=100 nM,N/P=10, RGD-PEG/ECO=2.5 mol %,) as previously described. ControlRGD-PEG-ECO/NC (NC) nanoparticles were similarly prepared usingsiLuciferase as the non-specific siRNA (NC). The targeted nanoparticlespossessed a narrow size and charge distribution, as characterized bydynamic light scattering (FIG. 16a,b ). The RGD-PEG-ECO/NC andRGD-PEG-ECO/siDANCR nanoparticles had a zeta potential of 20±4.6 mV and24.67±3.6 mV and hydrodynamic diameter of 107.83±19.28 nm and109.12±6.34 nm, respectively, FIG. 16c . The siRNA loading efficiencyand encapsulation in the nanoparticles were evaluated by gel retardationassay, FIG. 16d . Both the nanoparticles demonstrated efficient siRNAencapsulation, with negligible free siRNA bands. The structure andmorphology of the nanoparticles were visualized by transmission electronmicroscopy (FIG. 16e ) and consistent sizes (ca. 100 nm) were observed.

The efficiency and kinetics of DANCR silencing by RGD-PEG-ECO/siDANCRnanoparticles were tested in MDA-MB-231 and BT549 cells with high DANCRexpression on days 1, 4, and 7 post-treatment. Compared to the control,RGD-PEG-ECO/siDANCR nanoparticles induced 80-90% silencing of DANCR inboth the cell lines for at least 7 days after a single transfection(FIG. 16f ). These results demonstrate that RGD-PEG-ECO/siDANCRnanoparticles can facilitate efficient, robust, and prolonged silencingof DANCR in TNBC cells.

RGD-PEG-ECO/siDANCR Nanoparticles Suppress the Invasion andProliferation of TNBC Cells

To determine if DANCR depletion plays a role in TNBC suppression, weperformed loss-of-function studies in MDA-MB-231 and BT549 cells treatedwith RGD-PEG-ECO/siDANCR and RGD-PEG-ECO/NC to evaluate functionalchanges. After confirming ˜80% knockdown of DANCR expression after 24 hof siDANCR-nanoparticle treatment (FIG. 17a ), the transfected cellswere tested for their migratory and invasive capacity. TheRGD-PEG-ECO/siDANCR-treated cells showed a significant reduction intheir ability to invade Matrigel-coated membranes in standard transwellassays compared to the control-treated cells, evidenced by the reducednumber of migrated cells stained using crystal violet (FIG. 17b ) andcounted using ImageJ (FIG. 17c ). The siDANCR-treated cells also showeda significant decrease in their ability to close the wounds scratchedinto a confluent monolayer, as compared to those treated with thecontrol (FIG. 17d ). These results demonstrate that DANCR downregulationinhibits the invasion and migration of TNBC cells.

Next, we determined the effect of RGD-PEG-ECO/siDANCR-mediated DANCRsilencing on the survival, growth, and proliferative abilities ofMDA-MB-231 and BT549 cells. The cells were treated with thenanoparticles for 48 h, and their viability was measured using the MTTassay. Compared to control, DANCR knockdown caused a significantdecrease (30-70%) in the viability of both the cell lines (FIG. 17e ),which was further validated by BrdU incorporation assay.RGD-PEG-ECO/siDANCR-treated cells showed significant reduction in BrdUincorporation (FIG. 17f ), indicating that DANCR knockdown suppressesthe proliferation of TNBC cells.

The effect of RGD-PEG-ECO/siDANCR treatment in MDA-MB-231 and BT549cells was also assessed in 3D Matrigel culture. Silencing of DANCRsignificantly reduced the ability of the cells to form 3D tumorspheroids, as compared to control-treated cells (FIG. 17g ), at 5 dayspost-treatment. Finally, the ability of single TNBC cells to grow andproliferate into colonies following DANCR silencing was evaluated bystandard clonogenic assay. Control and siDANCR-treated cells wereserially-diluted and allowed to grow for 10-15 days. Individual coloniesformed were stained with 0.01% crystal violet and counted. As shown inFIG. 17h and FIG. 21, RGD-PEG-ECO/siDANCR-treated cells showedsignificant reduction of colonies than the NC-treated cells, indicatingthat loss of DANCR can inhibit the ability of TNBC cells to establishnew colonies.

RGD-PEG-ECO/siDANCR Nanoparticles Suppress TNBC Invasion andProliferation by Influencing Multiple Oncogenic Targets

The mechanism of biological functions of DANCR was investigated byevaluating the expression of multiple oncoproteins in MDA-MB-231 andBT549 cells treated with RGD-PEG-ECO/siDANCR or ECO-PEG-RGD/NC. Thesecell lines represent the claudin-low mesenchymal TNBC subtype, which isparticularly aggressive, and is characterized by highly active EMT andWnt signaling. As shown in FIG. 18a , DANCR silencing downregulated theexpression of the Wnt signaling protein 0-catenin, the EMT markers ZEB 1and N-cadherin, as well as the anti-apoptotic marker survivin. DANCR isknown to mediate its effects through EZH2-mediated epigeneticregulation. Since EZH2 is a part of Polycomb repressive complex 2 (PRC2)that facilitates target gene silencing through trimethylation of H3K27residues, we tested the expression of EZH2 and SUZ12 in siDANCR-treatedcells and found a significant decrease in the expression of the twoproteins. This was also accompanied by a reduction in the total levelsof H3K27-trimethylation (FIG. 18b ), suggesting that DANCR may play arole in PRC2-mediated trimethylation and repression of target genes.

Since siDANCR strongly inhibits PRC2 expression, we performed RNAimmunoprecipitation (RIP) for EZH2. As shown in FIG. 18c , DANCRdirectly binds to EZH2 in both MDA-MB-231 and BT549 cells, showing a12-fold and 8-fold enrichment with anti-EZH2 antibody pull-down,respectively. This enrichment was lost when the cells were treated anEZH2-inhibitor, DZNep. This direct binding coupled with the methylationchanges suggests that DANCR influences the expression of its targetgenes by epigenetic control.

Because lncRNAs are known to play dynamic roles in multiple oncogenicsignaling pathways, we evaluated the effect of DANCR silencing onmolecular alterations in the TNBC phosphorylation network using aPhospho-kinase array. The functional status of most oncogenes and tumorsuppressors is governed by post-translational modifications likephosphorylation, which are widely dysregulated in TNBC. Among these, thephosphorylation status of over 40 kinases expressed in MDA-MB-231 andBT549 cells were analyzed after treatment with RGD-PEG-ECO/siDANCR andRGD-PEG-ECO/NC, FIG. 18d . The kinases that showed a significant changein phosphorylation with siDANCR treatment are highlighted by numberedboxes and depicted in FIG. 18e . A common subset of tumor-promotingkinases, including p38α, ERK1/2, AMPKα1, Src, RSK, FAK, and PRAS40(#1-7), was significantly less phosphorylated with DANCR silencing inboth cell lines.

Interestingly, DANCR knockdown showed a differential phosphorylationpattern between the 2 TNBC cell lines, with reduced phosphorylation of 2distinct residues of AKT1/2/3 (T308 in MDA-MB-231 vs S473 in BT549cells, #11), contrasting S133 phosphorylation levels of CREB (#9),phosphorylation of JNK1/2/3 and PDGF-Rβ in MDA-MB-231 (#12,13) only, andphosphorylation of Fyn, Yes, WNK1, and PYK in BT549 (#12-15) only. DANCRsilencing reduced the total level of β-catenin (*) in both cell lines,independently validating the results in FIG. 18a , and also altered thephosphorylation patterns of tumor suppressors like p53, p²⁷ andtranscription factors (TFs) like Stat proteins. The endogenous kinaseprofiles and the siDANCR-mediated changes in these profiles are distinctbetween the two cell lines, highlighting the heterogeneity of thedisease and broad functions of the lncRNA. Our results underscore thedynamic and complex role of DANCR in impacting multiple oncogenic andtumor suppressor proteins in different TNBC models.

Systemic Administration of RGD-PEG-ECO/siDANCR Nanoparticles MediatesEffective Therapy of TNBC In Vivo

The efficacy of RGD-PEG-ECO/siDANCR nanoparticles in treating TNBC wasinvestigated in mice bearing MDA-MB-231 and BT549 xenografts. For the invivo experiments, RGD-PEG-ECO/siDANCR and RGD-PEG-ECO/NC nanoparticleswere formulated at N/P=8, RGD-PEG/ECO=2.5 mol %, and siRNA dose=1.0mg/kg. Given the higher siRNA concentration, the nanoparticles werelarger than those formed for in vitro transfections (FIG. 16), (averagediameter 189.46±14.3 nm and 188.11±6.82 nm for RGD-PEG-ECO/NC andRGD-PEG-ECO/siDANCR nanoparticles, respectively) (FIG. 19a ). Comparedto unmodified ECO/NC and ECO/siDANCR nanoparticles (51.15±2.15 mV and53.1±1.7 mV), the RGD-PEG-ECO/NC and RGD-PEG-ECO/siDANCR nanoparticlesshowed a reduction in the zeta potential (29.7±0.6 mV and 40.35±1.65 mV,respectively), indicating proper RGD-PEG conjugation (FIG. 19b ). Micewere intravenously injected with RGD-PEG-ECO/NC and RGD-PEG-ECO/siDANCRnanoparticles weekly for 6 weeks. The treatment with RGD-PEG-ECO/siDANCRresulted in a complete suppression of tumor proliferation and reducedtumor volumes (90.3±18.77 to 59.79±18.73 mm³ for MDA-MB-231 and72.9±14.75 to 58.79±18 mm³ for BT549), while the RGD-PEG-ECO/NC-treatedmice showed a rapid tumor growth (91.5±32 to 718±237 mm³ for MDA-MB-231and 77.77±17.4 to 868.1±238.3 mm³ for BT549), FIG. 19c-e and FIG. 22.

H&E staining of the tumors showed poorly differentiated high-grademalignancy (FIG. 19f ). While the NC-treated tumors showed large areasof zonal necrosis, the siDANCR-treated tumor sections showed only focalareas of necrosis. The efficacy of RGD-PEG-ECO/siDANCR was alsovalidated by measuring DANCR expression in total RNA extracted from theprimary tumors. Significant downregulation of DANCR was observed in thesiDANCR-treated tumors, compared to the NC-treated tumors (FIG. 19g ),confirming that RGD-PEG-ECO/siDANCR effectively delivered siDANCR intothe cancer cells to mediate efficient DANCR silencing and anti-tumoractivity.

As quantified by qRT-PCR, significant reduction of EZH2 (FIG. 19h ) andsurvivin (FIG. 19i ) was observed in the siDANCR-treated tumors,suggesting that the tumor suppression and regression are likely due toepigenetic changes and apoptotic/cytostatic effects of systemic siDANCRtherapy.

No changes in the body weight were observed in the siDANCR- andNC-treated mice during the entire treatment period (FIG. 20a ). Thevital organs of the mice were assessed by H&E-staining. As shown in FIG.20b-d , the liver, kidneys, and lungs showed normal hepatocyte, renal,and alveolar histology, with no evidence of inflammation andfibrosis/interstitial disease, respectively. No significant differencein morphology was observed between the siDANCR- and NC-treated mice,indicating that repeated injections of the nanoparticles did notadversely affect the vital organs and health of the mice.

ECO self-assembles with siRNA to form stable ECO/siRNA nanoparticles,which possess unique features that protect the siRNA cargo duringsystemic delivery and facilitate pH-sensitive endosomal escape andreductive cytosolic siRNA release (PERC) in target cells.RGD-PEG-ECO/siDANCR nanoparticles mediate robust silencing of DANCR inTNBC cells for at least a week. DANCR knockdown inducesapoptotic/cytostatic effects and inhibits invasion, migration, andcolony formation, which are important characteristics of tumorigenesis,proliferation, and aggression in TNBC. Consequently, weekly systemicinjections of RGD-PEG-ECO/siDANCR nanoparticles at a low dose result intumor suppression and regression in TNBC models. It can be speculatedthat the tumor growth arrest or cytostasis may, in part, be due toincreased apoptosis, suppression of tumor-promoting proteins, and as yetunidentified epigenetic changes. The therapeutic efficacy of DANCRsilencing can be attributed to the diverse gene expression changes weobserved in the treated TNBC cells.

Although the overexpression of DANCR has been shown to be associatedwith poor prognosis and tumor progression in multiple cancers, little isknown about the pathways that upregulate DANCR in TNBC. During epidermaland osteoblast development, DANCR is required to maintain the progenitorcells in the undifferentiated state. Since TNBC consists of many poorlydifferentiated cell types, and a high content of cancer stem cells, itis possible that the maintenance of self-renewal and cancer sternness orthe transformation of well-differentiated mammary epithelial cells intothe highly aggressive, mesenchymal and poorly differentiated phenotyperequires or triggers the overexpression of DANCR. Irrespective of theunderlying causes, we demonstrate that DANCR functions pleiotropicallyat epigenetic, transcriptional, translational, and post-translationallevels of gene expression. DANCR silencing results in inhibition of EZH2and PRC2-mediated H3K27-trimethylation at the epigenetic level;downregulation of TFs β-catenin, ZEB 1, and Stat proteins, and proteinsN-cad and survivin; and decreased phosphorylation of Src, FAK, and otheroncoproteins. These proteins are upregulated in TNBC; they play criticalroles in disease progression, metastasis and drug resistance, and someof them are common targets for developing targeted therapies.Unfortunately, limited curative outcomes have been achieved with thesetargeted therapies individually. The pleiotropic effects of DANCRsilencing with RGD-PEG-ECO/siDANCR may overcome the limitations ofconventional approaches of targeting an individual pathway or protein toachieve better therapeutic outcomes. DANCR silencing and the consequentdownregulation of the EZH2-SUZ12 axis of the PRC2 complex likely altersthe recruitment or binding of EZH2 to its target genes or proteins, inturn impacting the transcriptional silencing of tumor promoters oractivation of tumor suppressors. It can be hypothesized that DANCRdownregulation enables collective repression of multiple TNBC-promotingpathways, including Wnt and EMT signaling, anti-apoptosis,phosphorylated FAK, Src, RSK1/2/3, AKT1/2/3, and other oncoproteins,resulting in efficacious therapeutic outcome. Thus, it can be speculatedthat the functional downregulation of a single onco-lncRNA cansimultaneously impair several different tumor-promoting pathways,potentially circumventing the problem of compensatory mitogenic pathwaysarising from drug resistance.

In summary, we demonstrate that DANCR is overexpressed in TNBC and playspleiotropic roles by regulating multiple molecular pathways. DANCRsilencing mediates effective regulation of an array of oncogenicactivities and inhibits invasion and proliferation of TNBC cells.Additionally, systemic treatment with targeted nanoparticles results intumor suppression and regression in two independent TNBC models.

Example 4

BANCR Summary

BANCR Expression in Drug-Resistant Breast Cancer and Prostate CancerCells

The lncRNA BRAF activated non-coding RNA (BANCR), a 4-exon transcript of693-bp, was first discovered as an oncogenic long non-coding RNA inBRAF^(V600E) melanomas cells in 2012 and was related to melanoma cellmigration. Besides melanoma, increasing evidence has explored thepotential role of BANCR in the development and progression of multipleother human malignancies, such as breast cancer and prostate cancer,etc. Here, we checked the expression of BANCR in breast cancer cell line(MCF-7) and its corresponding palbociclib-resistant cell line (MCF-7-DR)and prostate cancer cell line (DU145) and its correspondingpaclitaxel-resistant cell line (DU145-DR) by RT-PCR. It was found thatboth MCF-7-DR and DU145-DR showed upregulated BANCR expression ascompared to their parental cells (FIG. 23).

ECO/siBANCR Nanoparticles Facilitate Efficient BANCR Silencing

To effectively downregulate BANCR expression in MCF-7-DR and DU145-DRcells, we formulated stable nanoparticles using the lipid ECO andsiBANCR via self-assembly. AllStars Negative Control siRNA (Qiagen) wasemployed as non-specific siRNA duplex (siNS). The BANCR-specific siRNAduplex (siBANCR, sense: 5′-GCA CAG GAC UCC AUG GCA AUU-3′ (SEQ ID NO:24); antisense: 5′-UUG CCA UGG AGU CCU GUG CUU-3′ (SEQ ID NO: 25)) wasalso purchased from Dharmacon. The particle size and zeta potentialdistribution of formulated ECO/siRNA were determined by dynamic lightscattering (DLS) measurement. Both ECO/siBANCR and ECO/siNS (control)nanoparticles displayed uniform size distribution with an averagehydrodynamic diameter of 169 nm and 155 nm (FIG. 24a ) and positive zetapotential of 33±0.8 mV and 29±1 mV, respectively (FIG. 24b ). Fortransfection, the plated MCF-7-DR and DU145-DR cells were treated withECO/siNS or ECO/siBANCR nanoparticles for 24 h. Thereafter, theefficiency of BANCR silencing by ECO/siBANCR nanoparticles were testedin MCF-7-DR and DU145-DR cells with high BANCR expression. Compared tothe control, ECO/siBANCR nanoparticles induced significant knockdown ofBANCR in both the cell lines (FIG. 24c ). These results demonstrate thatECO/siBANCR nanoparticles can facilitate efficient silencing of BANCR.

Example 5

HOTTIP Summary

HOTTIP and HOXA13 Expression in Prostate Cancer (PCa) Cells

The lncRNA HOXA transcript at the distal tip (HOTTIP) located at the5′-end of the HOXA cluster, which is a key locus control element of HOXAgenes, and is brought into close proximity to the 5′ HOXA genes bychromosomal looping. HOTTIP has been suggested as a potential prognosticbiomarker and therapeutic target for diverse cancers. HOTTIP plays majorroles in cell proliferation, EMT, and metastasis, by regulating HOXA13.Here, we firstly checked the expression of HOTTIP and HOXA13 indifferent PCa cell lines by RT-PCR, DU145 and its correspondingpaclitaxel (PTX)-resistant cell line, DU145-DR, PC3 and itscorresponding PTX-resistant cell line, PC3-DR, and C4-2. As shown inFIG. 25, it was found that both HOTTIP (FIG. 25a ) and HOXA13 (FIG. 25b) were significantly upregulated in PC3 and DU145-DR cells.Interestingly, the expression of HOTTIP and HOXA13 exhibited a highcorrelation score in PCa cells (Person's r=0.993, FIG. 25c ). We alsodetermined the expression level of HOTTIP and HOXA13 in 496 primarytumor tissues samples (data from TCGA). Consistently, the expression ofHOTTIP and HOXA13 also had a strongly positive correlation in PCatissues (Person's r=0.715, FIG. 26).

Considering the high expression of HOTTIP in PC3 and DU145-DR cells, wechose these two cell lines as models to study the role of HOTTIP in PCacells. According to our previous study, PC3 and DU145-DR cells bothexhibit an elongated and spindle-like morphology, which ischaracteristic of mesenchymal cells (FIG. 27).

ECO/siHOTTIP Nanoparticles Facilitate Efficient HOTTIP Silencing in PCaCells

To effectively downregulate HOTTIP expression in PCa cells, weformulated stable nanoparticles using the lipid ECO and siHOTTIP viaself-assembly. The pH-sensitive lipid ECO was synthesized in our lab. A5 mM stock solution of ECO (MW: 1023) in ethanol was employed to formnanoparticles with siRNA. The siRNA was dissolved in RNase-free water ata concentration of 25 μM. ECO/siRNA nanoparticles were prepared bymixing the stock solutions of ECO and siRNA at an N/P ratio of 10 inRNase-free water under gentle agitation for about half an hour. TheLincode Non-Targeting Control siRNA #1 (Dharmacon) was employed asnon-specific siRNA duplex (siNS). The HOTTIP-specific siRNA duplex(siHOTTIP, sense: 5′-CGG CAG GAG CCC AAG GAA AUU-3′ (SEQ ID NO: 26);antisense: 5′-UUU CCU UGG GCU CCU GCC GUU-3′ (SEQ ID NO: 27)) was alsopurchased from Dharmacon. ECO/siRNA nanoparticles have been demonstratedto efficiently mediate cytosolic siRNA delivery and gene silencingthrough pH-sensitive amphiphilic endosomal escape. The particle size andzeta potential distribution were determined by dynamic light scattering(DLS) measurement. Both ECO/siHOTTIP and ECO/siNS (control)nanoparticles displayed uniform size distribution with an averagehydrodynamic diameter of 111 nm and 95 nm (FIG. 28a ) and positive zetapotential of 32±1 mV and 45±1 mV, respectively (FIG. 28b ). The abilityof ECO to entrap siRNA was evaluated by gel electrophoresis. ECO/siRNAnanoparticle solution (7 μL) mixed with 3 μL of loading dye was loadedinto a 1% agarose gel. Free siRNA was also loaded as control. Afterelectrophoresis at 100 V for 20 min, the gel was imaged using a ChemiDocXRS+ System to visualize the encapsulated and free siRNA bands. The gelretardation assay demonstrated efficient encapsulation of siRNA in thenanoparticles, with negligible free siRNA bands (FIG. 28c ). Theseresults indicate that stable ECO/siRNA nanoparticle formulations with arobust entrapment of negatively charged siRNA were achieved.

For transfection, the plated PC3 and DU145-DR cells were treated withECO/siNS or ECO/siHOTTIP nanoparticles for 24 h. Thereafter, theefficiency of HOTTIP silencing by ECO/siHOTTIP nanoparticles were testedin PC3 and DU145-DR cells with high HOTTIP expression. Compared to thecontrol, ECO/siHOTTIP nanoparticles induced significant knockdown ofHOTTIP in both the cell lines (FIG. 28d ). These results demonstratethat ECO/siHOTTIP nanoparticles can facilitate efficient silencing ofHOTTIP in PCa cells.

ECO/siHOTTIP Inhibits Proliferation of PCa Cells

In order to study the effect of ECO/siHOTTIP in proliferation of PCacells. We performed a cell viability assay using CCK-8. PCa cells(untreated, UT) or PCa cells transfected with ECO/siRNA nanoparticles(siNS or siHOTTIP) were seeded onto a 96-well plate at a density of5,000 cells/well and incubated overnight to allow adherence. CCK-8 assaywas performed at day 1, 2 and 3 after seeding according tomanufacturer's protocol. Briefly, CCK-8 with a 10% vol. of the mediumwas added into each well. After 1 h incubation at 37° C., the absorbance(O.D.) of the solution at 450 nm was measured using a microplate reader.The O.D. values were normalized to the blank. The experiments werecarried out in quadruplicate.

As shown in FIG. 29, compared to untreated group and siNS control,HOTTIP knockdown caused a significant decrease in the viability of boththe cell lines at day 1 to day 3, indicating that HOTTIP knockdownsuppresses the proliferation of PCa cells.

This was further validated by live/dead staining using calcein-AM(Dojindo) and propidiumiodide (PI). Approximately 1,000,000 PCa cells(untreated, UT) or PCa cells transfected with ECO/siRNA nanoparticles(siNS or siHOTTIP were seeded onto a 6-well plate for 24 h. At day 3after seeding, the cells were washed by pre-warmed PBS and stained with2 μM calcien-AM for live cells (in green fluorescence) and 4 μM PI fordead cells (in red fluorescence) for 15 min at 37° C. Thereafter, thesamples were imaged under a confocal microscope. As shown in FIG. 30,compared to untreated group and siNS control, HOTTIP knockdown caused asignificant increase in the number of dead cells (in red fluorescence).Moreover, the effect of silencing HOTTIP by ECO/siHOTTIP nanoparticleson cell viability were dose dependent. As increase in the dose ofsiHOTTIP, the cell viability significantly decreased compared to thesiNS control in PC3 cells (FIG. 31).

3D tumor spheroid growth assay, a cell culture technique that provides amore physiological model to mimic in vivo tumor conditions wasperformed. Transfected PCa cells (300,000) were suspended in 5%Matrigel-containing media and plated on to 24-well plates coated with athick layer of Corning™ Matrigel™ Membrane Matrix. The ability of thecells to form tumor spheroids in the 3D Matrix was monitored andphotographed at day 7 using the Moticam T2 camera. Silencing of HOTTIPsignificantly reduced the ability of the cells to form 3D tumorspheroids, as compared to siNS control-treated cells (FIG. 32), at 7days' post-treatment. Taken together, these results indicate thatsilencing HOTTIP by ECO/siRNA nanoparticles can inhibit proliferationand 3D tumor spheroid growth of PCa cells (PC3 and DU145-DR).

ECO/siHOTTIP Inhibits Invasion, Migration and Epithelial-MesenchymalTransition (EMT) of PCa Cells

The transfected cells were tested for their migratory and invasivecapacity by scratch wound closure assay and transwell cell migration andinvasion assay. For scratch wound closure assay, approximately 1,000,000PCa cells transfected with ECO/siRNA nanoparticles were seeded onto a6-well plate for 24 h. A sterilized 200 μL pipette tip was used to pressfirmly against the top of the plate and make a vertical wound downthrough the cell monolayer. After aspirating the media and cell debris,fresh media was added slowly to avoid detaching additional cells. Aninitial 0 h picture was taken using Moticam and wound closure wasmonitored for up to 24 h post-scratch. The siHOTTIP-treated cells alsoshowed a significant decrease in their ability to close the woundsscratched into a confluent monolayer, as compared to those treated withthe control (FIG. 33).

For transwell cell migration and invasion assay, PCa cells transfectedwith ECO/siRNA nanoparticles were seeded onto Transwell inserts coatedwith (for invasion) or without (for migration) 0.28 mg/mL Corning™Matrigel™ Membrane Matrix (Corning, USA). After 24 h, cotton-tippedapplicators were used to remove the gel and/or remaining cells that havenot migrated from the top of the inserts. Then, the inserts were fixedwith 3.7% formalin for 10 min and immersed in 0.1% crystal violet for 20min to stain the migrated cells. Images of the stained inserts weretaken using Moticam. ECO/siHOTTIP treated PC3 and DU145-DR cells showeda significant reduction in their ability of migration and invasion,evidenced by the significantly reduced number of migrated cells stainedin purple compared with those treated with ECO/siNS (FIG. 34).

In addition to functional changes, silencing of HOTTIP with ECO/siHOTTIPnanoparticles also resulted in changes in molecular signatures of PC3and DU145-DR cells. Reduced expression of the mesenchymal markers,ZEB-1, N-cadherin, β-Catenin and vimentin, was observed in the westernblots of ECO/siHOTTIP treated cells (FIG. 35). Taken together, theseresults indicate that silencing HOTTIP by ECO/siRNA nanoparticles caninhibit invasion, migration and EMT of PCa cells (PC3 and DU145-DR).

Role of HOTTIP in TGF-β1 Treated DU145 Cells

In order to confirm the role of HOTTIP in PCa cells with mesenchymalfeature, DU145 cells, which display the epithelial hallmark of denselypacked squamous morphology and express lower level of HOTTIP, weretreated with 5 ng/mL TGF-β1. After treated with TGF-β1, DU145-TGFβ1cells exhibited mesenchymal hallmark of elongated and spindle-likemorphology. Furthermore, significant upregulation of mesenchymalmarkers, N-cadherin, and downregulation of epithelial marker, E-cadherinwas observed for the DU145-TGF-β1 cells as compared to DU-145 cells.Interestingly, expression of HOTTIP was also upregulated after treatmentwith TGF-β1 (FIG. 36).

Next, we determined the effect of ECO/siHOTTIP-mediated HOTTIP silencingon the migration and invasion capacity of DU145-TGF-β1 cells usingTranswell study. Similar to the result of PC3 and DU145-DR cells,ECO/siHOTTIP treated DU145-TGF-β1 cells showed a significant reductionin their ability of migration and invasion, evidenced by thesignificantly reduced number of migrated cells stained in purplecompared with those treated with ECO/siNS (FIG. 37).

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All references,publications, and patents cited in the present application are hereinincorporated by reference in their entirety.

Having described the invention, we claim:
 1. A nanosized complexcomprising siRNA and a compound comprising formula (I):

wherein R¹ is an alkylamino group or a group containing at least onearomatic group; R² and R³ are independently an aliphatic group or ahydrophobic group; R⁴ and R⁵ are independently H, a substituted orunsubstituted akyl alkyl group, an alkenyl group, an acyl group, anaromatic group, polymer, a targeting group, or a detectable moiety; a,b, c, and d are independently an integer from 1 to 10; andpharmaceutically acceptable salts thereof.
 2. The nanosized complex ofclaim 1, wherein R¹ comprises at least one of:

where R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are independentlyhydrogen, an alkyl group, a hydrophobic group, or a nitrogen containingsubstituent; and e, f, g, i, j, k, and l, are an integer from 1 to 10.3. The nanosized complex of claim 1, wherein R² and R³ are independentlya hydrophobic group derived from oleic acid or linoleic acid.
 4. Thenanosized complex of claim 3, wherein R² and R³ are the same.
 5. Thenanosized complex of claim 1, wherein R⁴ and R⁵ are independently H, asubstituted or unsubstituted polymer, a targeting group, or a detectablemoiety.
 6. The nanosized complex of claim 1, wherein a, b, c, and d areeach
 2. 7. The nanosized complex of claim 1, R¹ comprises CH₂CH₂NH₂. 8.The nanosized complex of claim 1, wherein the compound is


9. The nanosized complex of claim 1, wherein polyethylene glycol iscovalently attached to the compound.
 10. The nanosized complex of claim1, wherein the targeting group is covalently attached to the compound bya linker.
 11. The nanosized complex of claim 10, wherein the linkercomprises a polyamino acid group, a polyalkylene group, or apolyethylene glycol group.
 12. The nanosized complex of claim 10,wherein the targeting group comprises a peptide, a protein, an antibody,or an antibody fragment.
 13. The nanosized complex of claim 11, whereinthe linker comprises an acid labile bond.
 14. The nanosized complex ofclaim 1, wherein the siRNA inhibits expression of an lncRNA upondelivery of the nanosized complex to a cell.
 15. The nanosized complexof claim 14, wherein the lncRNA is an onco-lncRNA.
 16. A method oftreating cancer in a subject in need thereof, the method comprising:administering to the subject a nanosized complex comprising siRNA and acompound comprising formula (I):

wherein R¹ is CH₂CH₂NH₂; R² and R³ are independently an aliphatic groupor a hydrophobic group; R⁴ and R⁵ are independently H, a substituted orunsubstituted alkyl group, an alkenyl group, an acyl group, an aromaticgroup, polymer, a targeting group, or a detectable moiety; a, b, c, andd are independently an integer from 1 to 10; and pharmaceuticallyacceptable salts thereof.
 17. The method of claim 16, wherein R² and R³are independently a hydrophobic group derived from oleic acid orlinoleic acid.
 18. The method of claim 16, wherein the compound is


19. The method of claim 16, wherein the siRNA inhibits expression of anlncRNA upon delivery of the nanosized complex to a cell.
 20. The methodof claim 16, wherein the IncRNA is an onco-lncRNA.